U.S. patent number 11,142,751 [Application Number 16/811,733] was granted by the patent office on 2021-10-12 for crispr-cas system for clostridium genome engineering and recombinant strains produced thereof.
This patent grant is currently assigned to AUBURN UNIVERSITY. The grantee listed for this patent is AUBURN UNIVERSITY. Invention is credited to Yi Wang, Jie Zhang.
United States Patent |
11,142,751 |
Wang , et al. |
October 12, 2021 |
CRISPR-cas system for Clostridium genome engineering and
recombinant strains produced thereof
Abstract
A system for modifying the genome of Clostridium strains is
provided based on a modified endogenous CRISPR array. The
application also describes Clostridium strains modified for
enhanced butanol production wherein the modified strains are
produced using the novel CRISPR-Cas system.
Inventors: |
Wang; Yi (Auburn, AL),
Zhang; Jie (Auburn, AL) |
Applicant: |
Name |
City |
State |
Country |
Type |
AUBURN UNIVERSITY |
Auburn |
AL |
US |
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Assignee: |
AUBURN UNIVERSITY (Auburn,
AL)
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Family
ID: |
72335992 |
Appl.
No.: |
16/811,733 |
Filed: |
March 6, 2020 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200283746 A1 |
Sep 10, 2020 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62815198 |
Mar 7, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/63 (20130101); C12N 15/74 (20130101); C12N
15/102 (20130101); C12N 9/22 (20130101); C12P
7/16 (20130101); C12N 2310/20 (20170501); C12R
2001/145 (20210501); Y02E 50/10 (20130101); C12N
1/205 (20210501) |
Current International
Class: |
C12N
9/22 (20060101); C12N 15/63 (20060101); C12N
1/20 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2007/332240 |
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Jun 2008 |
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AU |
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2008/052973 |
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May 2008 |
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WO |
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2012/045022 |
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Apr 2012 |
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WO |
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2015/159086 |
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Oct 2015 |
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WO |
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2015159087 |
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Oct 2015 |
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WO |
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Other References
Jang et al., mBio, 3(5), 2012, pp. 1-9. cited by examiner .
Ou, et al, High butanol production by regulating carbon, redox and
energy in Clostridia, Front. Chem. Sci. Eng. 2015, 9(3): 317-323.
cited by applicant .
Pyne et al, Harnessing heterologous and endogenous CRISPR-Cas
machineries for efficient markerless genome editing in Clostridium,
Scientific Reports, May 2016, 1-15. cited by applicant .
Yu et al, Metabolic engineering of Clostridium tyrobutyricum for
n-butanol production, Metabolic Engineering 2011, 13(4), 373-382.
cited by applicant .
Keis, S., Shaheen, R., Jones, D. T., Emended descriptions of
Clostridium acetobutylicum and Clostridium beijerinckii, and
descriptions of Clostridium saccharoperbutylacetonicum sp. Nov. and
Clostridium saccharobutylicum sp. nov. International Journal of
Systematic and Evolutionary Microbiology 2001, 51, 2095-2103. cited
by applicant .
Lee, S. Y., Park, J. H., Jang, S. H., Nielsen, L. K., et al.,
Fermentative butanol production by clostridia. Biotechnology and
Bioengineering 2008, 101, 209-228. cited by applicant .
Lee, J., Jang, Y.-S., Han, M.-J., Kim, J. Y., Lee, S. Y.,
Deciphering Clostridium tyrobutyricum metabolism based on the
whole-genome sequence and proteome analyses. mBio 2016,
7(3):e00743-16. cited by applicant .
Zhang, J., Zong, W., Hong, W., Zhang, Z.-T., Wang, Y., Exploiting
endogenous CRISPR-Cas system for multiplex genome editing in
Clostridium tyrobutyricum and engineer the strain for high-level
butanol production. Metabolic Engineering 2018, 47, 49-59. cited by
applicant .
Lehmann, D., Honicke, D., Ehrenreich, A., Schmidt, M., et al.,
Modifying the product pattern of Clostridium acetobutylicum:
physiological effects of disrupting the acetate and acetone
formation pathways. Applied Microbiology and Biotechnology2012, 94,
743-754. cited by applicant .
Yu, M. R., Zhang, Y. L., Tang, I. C., Yang, S. T., Metabolic
engineering of Clostridium tyrobutyricum for n-butanol production.
Metabolic Engineering 2011, 13, 373-382. cited by
applicant.
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Primary Examiner: Monshipouri; Maryam
Attorney, Agent or Firm: Barnes & Thornburg LLP
Government Interests
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under Grant number
ALA014-1-15017 awarded by the US Department of Agriculture (USDA),
National Institute of Food and Agriculture (NIFA). The government
has certain rights in the invention.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to the following U.S. Provisional
Patent Application No. 62/815,198 filed Mar. 7, 2019. The
disclosure of which is hereby expressly incorporated by reference
in its entirety.
Claims
What is claimed is:
1. A Clostridium strain modified for enhanced butanol production
relative to wild type C. tyrobutyricum (ATCC 25755), said
Clostridium strain comprising a modification to the native cat1
gene, said modification preventing expression of a functional cat1
gene product; and an exogenous sequence encoding i) an aldehyde
dehydrogenase; ii) a bifunctional aldehyde/alcohol dehydrogenase;
or iii) an aldehyde dehydrogenase and an alcohol dehydrogenase.
2. The Clostridium strain of claim 1 wherein said Clostridium cat1
gene is modified by the insertion of said exogenous sequence into
the cat1 gene rendering the cat1 gene incapable of expressing a
functional gene product.
3. The Clostridium strain of claim 2 wherein said exogenous
sequences comprises a bifunctional alcohol/aldehyde dehydrogenase
gene selected from the group consisting of adhE1 and adhE2.
4. The Clostridium strain of claim 3 wherein said modified strain,
when cultured at a temperature of less than 30.degree. C. using
glucose as a carbon source, produces at least 20 g/L of butanol
after 72 hours of culture.
5. The strain of claim 1 wherein the strain is the Clostridium
tyrobutyricum strain deposited with Agriculture Research Service
Culture Collection (NRRL) and assigned accession no. NRRL
B-67519.
6. A method of producing butanol, said method comprising the steps
of culturing the Clostridium strain of claim 1 under conditions
suitable for growth of the strain; and recovering the butanol
produced by said cell.
7. The method of claim 6 wherein the strain is cultured at a
temperature selected from the range of about 20.degree. C. to about
30.degree. C.
8. A Clostridium strain modified for enhanced butanol production
relative to wild type C. tyrobutyricum (ATCC 25755), said
Clostridium strain being selected from the group consisting of
Clostridium butyricum, Clostridium thermobutyricum, Clostridium
cellulovorans, Clostridium carboxidivorans, Clostridium
tyrobutyricum, Clostridium polysaccharolyticum, Clostridium
populeti, and Clostridium kluyveri and comprising a modification to
the native cat1 gene, said modification preventing expression of a
functional cat1 gene product; and an adhE gene introduced into said
cell and having at least 95% sequence identity to a C.
acetobutylicum aldehyde/alcohol dehydrogenase gene of SEQ ID NO:
133 or SEQ ID NO: 134.
9. The Clostridium strain of claim 8 wherein said Clostridium cat1
gene is modified by the insertion of said adhE gene into the cat1
gene rendering the cat1 gene incapable of expressing a functional
gene product.
10. The Clostridium strain of claim 9 wherein said Clostridium
strain is Clostridium tyrobutyricum.
11. The Clostridium strain of claim 9 wherein the entire coding
region of said Clostridium cat1 gene is replaced with the inserted
adhE gene.
Description
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
Incorporated by reference in its entirety is a computer-readable
nucleotide/amino acid sequence listing submitted concurrently
herewith and identified as follows: a 40 kilobytes ACII (Text) file
named "314658ST25.txt" created on Feb. 20, 2020.
BACKGROUND
n-butanol (butanol hereafter) is used as a solvent, paint thinner,
perfume, and more recently as a source of renewable fuel. Hence,
methods to enhance butanol production are a major focus. However,
traditional chemical synthesis methods employed for butanol
production are costly and laborious. Furthermore, these methods
generate unwanted byproducts and environmental pollutants.
Alternative approaches continue to be investigated for their
ability to overcome these limitations while also significantly
increasing the yield of desired products, particularly butanol.
These alternatives include the use of microbial host strains that
can be exploited for their natural ability to produce butanol.
Clostridia are a type of bacteria that have long been studied for
biobutanol production through their acetone-butanol-ethanol (ABE)
fermentation pathway. Although large scale production has already
been established using clostridia, there are several obstacles that
prevent it from being economically feasible, including high costs
and low yields associated with batch fermentation of currently
available Clostridia strains.
Recent efforts have focused on modifying the ABE fermentation
pathway of clostridia in order to reduce unwanted byproducts while
increasing overall yield of butanol. One method used to achieve
these modifications involves the use of CRISPR-Cas9 systems which
have been widely used as a genome editing tool for numerous types
of bacteria. However, conventional CRISPR methods are limited by
severe toxicity to the host cells and thus in many cases are
difficult to implement. Hence, alternative strategies are needed to
improve butanol production while also overcoming existing
limitations.
Clustered regularly interspaced short palindromic repeats (CRISPR)
and the CRISPR-associated (Cas) system is an RNA guided immune
system in bacteria and archaea that can provide defense against
foreign invaders, such as phages and plasmids. Most currently
identified CRISPR-Cas systems share similar features, consisting of
identical direct repeats separated by variable spacers, along with
a suite of associated cas genes. CRISPR-Cas systems can be
classified into two classes and six types based on the signature
Cas proteins and the architecture of CRISPR-cas loci. A complex of
multiple Cas proteins are involved in degrading the invading
genetic elements in Types I, III and IV, which all belong to the
Class 1 system; while Types II, V and VI in the Class 2 system can
carry out the same operation by using a single large Cas protein.
Among the various CRISPR-Cas systems, Type I, II, and III are the
most widespread in both archaea and bacteria, and distinguished by
the presence of the unique signature protein: Cas3, Cas9, and
Cas10, respectively. Among them, Type I systems exhibit the most
diversity, and are further divided into six subtypes: I-A to
I-F.
Three functional stages, termed adaptation, expression, and
interference, are generally included in the development of the
immunity of CRISPR-Cas systems for the defense of the potential
foreign invaders. During the adaptation phase, spacer sequences
derived from the invading genetic elements are identified and
integrated into the host genome right between the leader sequence
and the first spacer, generating the new spacers of the CRISPR
array. A promoter located within the CRISPR leader sequence then
drives the transcription of CRISPR array (including the new
spacers) to form a long precursor CRISPR RNA (crRNA) followed by
the cleavage of the precursor crRNAs to make mature crRNAs. Once
the invasion happens again to the host cells, a ribonucleoprotein
complex (crRNP) will be formed by the mature crRNAs and specific
Cas proteins to recognize the same or similar foreign genetic
elements though sequence matching between the spacer on the crRNA
and the protospacer on the foreign invaders, and degrade the
invading DNA or RNA via interference. During the interference in
Type I and Type II systems, the targeting efficiency is greatly
improved if the protospacer is flanked by a short conserved
sequence defined as protospacer-adjacent motif (PAM). The PAM
sequence is usually 2-5 nucleotides long and located at the 5'- or
3'-end of the protospacer. The presence of PAM sequence in the
target DNA rather than in the CRISPR array of the host genome is
used to discriminate `self` and `non-self`.
Although the Class 2 system is less abundant in the nature, their
acting machineries are much simpler and more programmable. In the
past few years, the Streptococcus pyogenes CRISPR-Cas9
(spCRISPR-Cas9) system has been engineered to be a high efficient
genome editing tool that has been implemented in a broad range of
organisms, such as bacteria, yeast, plants, mammal cells, and human
cells. Besides single gene knock-in or knock-out, successes have
also been reported for multiplex genome editing and transcriptional
regulation, including repression and activation. Recently, another
Class 2 CRISPR effector, Cpf1, was characterized and repurposed for
genome editing. Compared to the CRISPR-Cas9 system, the CRISPR-Cpf1
system exhibited higher targeting efficiency and capability under
particular circumstances.
CRISPR-Cas9/Cpf1 systems have proven to be powerful genome
engineering tools with which versatile genome editing purposes can
be achieved. However, as a heterologous protein, in many cases,
either Cas9 or Cpf1 is hard to introduce into bacteria and archaea
due to their intrinsic toxicity, leading to low transformation
efficiency and thus difficulty for genome editing.
It has been reported that, based on genome analysis, approximately
47% of sequenced bacteria and 87% of sequenced archaea harbor
CRISPR-cas loci. Therefore, endogenous CRISPR-Cas systems have the
potential to be repurposed for genome editing and transcriptional
regulation. Through the deletion of cas3 gene which is responsible
for degrading the target DNA, the endogenous Type I-E CRISPR-Cas
system in Escherichia coli was harnessed as a programmable gene
expression regulator. Pyne et al. engineered the Type I-B
CRISPR-Cas system in Clostridium pasteurianum to be an efficient
genome editing tool, and successfully deleted the cpaAIR gene (Pyne
et al., 2016, Sci. Rep. 6, 25666).
In recent years, the genus Clostridium has drawn tremendous
attentions as it contains various strains with great potentials for
the production of commodity chemicals and fuels, such as butanol.
Butanol can be naturally produced in solventogenic clostridia
through the Acetone-Butanol-Ethanol (ABE) fermentation. Although
tremendous efforts have been invested on the metabolic engineering
of solventogenic clostridial strains for enhanced biobutanol
production, only very limited success has been achieved. This is
because, on one hand, there are several intrinsic byproducts in ABE
fermentation including fatty acids, acetone and ethanol that are
hard to eliminate; on the other, the ABE fermentation for butanol
production goes through a biphasic process and is subjected to
complicated metabolic regulation.
Yu et al. engineered C. tyrobutyricum ATCC 25755 (a hyper-butyrate
producer) for butanol production by inactivating the native acetate
kinase (ack) gene or the phosphate++(ptb) gene and introducing the
aldehyde/alcohol dehydrogenase (adhE2) from C. acetobutylicum, to
generate a strain that produces a butanol titer of 10.0 g/L (Yu et
al., 2011, Metab. Eng. 13, 373-82). Recently, the
butyrate-producing metabolism of C. tyrobutyricum was further
elucidated through whole-genome sequencing and proteomic analysis.
Interestingly, contradictory with the results by Yu et al. (Yu et
al., 2011), it was demonstrated that the ptb gene actually does not
exist in C. tyrobutyricum and the ack gene can't be deleted because
the deletion would lead to no end product and inefficient ATP
generation. Additionally, it was revealed that the butyrate
production in C. tyrobutyricum is in fact dependent on the
butyrate:acetate CoA transferase gene (cat1), which is very
different from the ptb-butyrate kinase (buk) pathway for butyrate
production in solventogenic clostridial strains. However, the
disruption of cat1 using mobile group II intron was unsuccessful,
because the inactivation of cat1 would likely lead to the inability
of the strain to carry out NADH oxidization.
Accordingly a need still exists for a bacterial strain that has
high levels of butanol production with decreased levels of
undesirable by products such as fatty acids and acetone. Applicants
provide herein a modified endogenous C. tyrobutyricum CRISPR-Cas
system under the control of an inducible promoter for modifying the
genome of clostridia. This system was used to generate a modified
C. tyrobutyricum that produces at least 20 g/L of butanol after 72
hours in a standard batch fermentation process.
SUMMARY
As disclosed herein, an efficient genome editing tool for C.
tyrobutyricum, is provided, based on the endogenous Type I-B
CRISPR-Cas system. The PAM sequences for DNA targeting purposes
were identified through in silico CRISPR array analysis and in vivo
plasmid interference assays. By using a lactose inducible promoter
to drive the transcription of the CRISPR array, multiplex genome
engineering purposes have been achieved, with an editing efficiency
as high as 100%.
In accordance with one embodiment a method of editing a bacterial
genome is provided wherein the method utilizes an endogenous
CRISPR-Cas system. One component of the system is a synthetic
CRISPR array that is optionally expressed under the control of an
inducible promoter. The CRISPR array encodes a spacer RNA that
targets a protospacer sequence contained within the bacterial
genome. The encoded array in conjunction with the native
Clostridium Cas protein forms a complex that will cleave the
targeted DNA. In one embodiment the method comprises introducing an
exogenous nucleic acid into the bacterial cell wherein the
exogenous nucleic acid comprises a sequence that encodes a
synthetic CRISPR array that is operably linked to an inducible
promoter, and optionally the exogenous nucleic acid further
comprises nucleic acid sequences that are homologous to sequences
flanking the target protospacer sequence to facillitate the
modification of the target genome loci through homologous
recombination.
In accordance with one embodiment the endogenous CRISPR-Cas system
of C. tyrobutyricum, was used to successfully engineer C.
tyrobutyricum for enhanced butanol production. By introducing an
adhE2 gene and inactivating the native cat1 gene, the obtained
mutant produced a record high of 26.2 g/L butanol in a batch
fermentation. This mutant bacterial strain of Clostridium
tyrobutyricum JZ100 was deposited in accordance with the provisions
of the Budapest Treaty on Nov. 5, 2017, with the Agriculture
Research Culture Collection (NRRL), an International Depository
Authority located at 1815 N. University Street, Peoria, Ill. 61604
and assigned accession number B-67519. This deposited strain can be
used as a robust workhorse for efficient biobutanol production from
low-value carbon sources, and can be further engineered for
enhanced butanol and other valuable biochemical production.
In accordance with one embodiment a vector for introducing
modifications into a target genomic site of bacteria, optionally a
Clostridium strain, via an endogenous CRISPR-Cas complex is
provided. In one embodiment the vector comprises a synthetic CRISPR
array, an inducible promoter operably linked to the synthetic
CRISPR array, and a first homology arm polylinker site. In one
embodiment the vector further comprises a native Clostridium
tyrobutyricum Cas encoding sequence. In one embodiment the
synthetic CRISPR array comprises a first spacer polylinker site, a
first and second direct repeat sequence, and a CRISPR terminator
sequence. In one embodiment the first and second direct repeat
sequence have greater than 95% sequence identity to one another, or
optionally, have 100% sequence identity to one another, and the
first spacer polylinker site is located between the first and
second direct repeat sequence.
In one embodiment a vector for introducing modifications into a
target genomic site of a Clostridium strain is provided wherein the
vector comprises a synthetic CRISPR array, a lactose inducible
promoter operably linked to the synthetic Type I-B CRISPR array, a
first homology arm polylinker site, and optionally a CRISPR
terminator sequence. In one embodiment the synthetic CRISPR array
comprises a first spacer polylinker site, and a first and second
direct repeat sequences, wherein the first and second direct repeat
sequences each comprise a sequence of SEQ ID NO: 2; and the first
spacer polylinker site located between the first and second direct
repeat sequences. In a further embodiment the CRISPR terminator
sequence comprises the sequence of SEQ ID NO 3.
In accordance with one embodiment a vector for multiplex
modification of a bacterial genome, optionally a Clostridium
strain, via a CRISPR-Cas complex is provided. In one embodiment the
vector comprises a synthetic CRISPR array, an inducible promoter
operably linked to the synthetic CRISPR array, a first homology arm
polylinker site and a second homology arm polylinker site. In one
embodiment the synthetic CRISPR array comprises a first spacer
polylinker site a second spacer polylinker site, and a first,
second and third direct repeat sequences, wherein the first, second
and third direct repeat sequences each have greater than 95%
sequence identity, or optionally at least 99% sequence identity to
the sequence of SEQ ID NO: 2, and the first spacer polylinker site
is located between the first and second direct repeat sequences and
the second spacer polylinker site located between the second and
third direct repeat sequences, and a CRISPR terminator sequence
located after the third direct repeat sequence.
In accordance with one embodiment a recombinant Clostridium strain
is provided that has been modified for enhanced butanol production.
In one embodiment, the Clostridium strain produces at least 20 g/L
of butanol after 72 hours of culture in a standard batch culture
procedure using glucose as the carbon source. In one embodiment the
modified Clostridium strain comprises an exogenous gene encoding
for aldehyde dehydrogenase activity, optionally wherein the
exogenous gene has been inserted into the native cat1 gene and
prevents expression of a functional cat1 gene product. In one
embodiment the exogenous aldehyde dehydrogenase gene is a dual
aldehyde/alcohol dehydrogenase gene including for example a C.
acetobutylicum gene selected from the group consisting of adhE1 and
adhE2. In one embodiment the recombinant Clostridium strain is
selected from the group consisting of Clostridium butyricum,
Clostridium thermobutyricum, Clostridium cellulovorans, Clostridium
carboxidivorans, Clostridium tyrobutyricum, Clostridium
polysaccharolyticum, Clostridium populeti, and Clostridium
kluyveri. In one embodiment the Clostridium strain is Clostridium
tyrobutyricum.
In one embodiment a method of biosynthetically producing butanol is
provided, wherein a modified Clostridium strain is cultured under
conditions suitable for growth of the strain, and the butanol
produce by the cell is recovered. In one embodiment the modified
Clostridium strain comprises a modification to the native cat1 gene
(wherein the modification inhibits or prevents expression of a
functional cat1 gene product); and an exogenous aldehyde
dehydrogenase gene, optionally wherein the aldehyde dehydrogenase
gene is inserted in to the genome of the Clostridium strain.
Optionally the exogenous aldehyde dehydrogenase gene encodes a
polypeptide having alcohol dehydrogenase and aldehyde dehydrogenase
activity. In one embodiment the exogenous aldehyde dehydrogenase
gene is selected from the group consisting of adhE1 and adhE2,
optionally wherein the adhE1 gene encodes a polypeptide having at
least 95% sequence identity to the polypeptide of SEQ ID NO: 133
and the adhE2 gene encodes a polypeptide having at least 95%
sequence identity to the polypeptide of SEQ ID NO: 134. In
accordance with one embodiment the Clostridium strain comprises a
cat1 gene modified by the insertion of an adhE1 or adhE2 gene into
the cat1 gene, rendering the cat1 gene incapable of expressing a
functional gene product. In one embodiment the culturing step
comprises culturing the modified Clostridium strain at a
temperature less than 37.degree. C., optionally at a temperature
selected from the range of about 20.degree. C. to about 30.degree.
C.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A & 1B Characterization of the Type I-B CRISPR-Cas
system in C. tyrobutyricum. FIG. 1A is a schematic diagram showing
the structure of the central Type I-B CRISPR-Cas locus in the
genome of C. tyrobutyricum. The central CRISPR-Cas locus possesses
a representative Type I-B cas operon including
cas6-cas8b-cas7-cas5-cas3-cas4-cas1-cas2 (labeled "cas68b753412")
followed by a leader sequence and the Array2 containing 8 distinct
spacers (diamonds) separated by 30-nt direct repeats (rectangles)
and a CRISPR terminator sequence (open circle). The transcription
of Array2 is driven by a promoter within the leader sequence. FIG.
1B provides sequence assignments providing an identification of
putative protospacer matches via in silico analysis of C.
tyrobutyricum CRISPR spacers. Only five nt of the 5'- and 3'-end
adjacent sequences are provided. Array1-17 (SEQ ID NO: 19); C.
themocellum ATCC 27405 (SEQ ID NO: 20) and Geobacillus sp. Y4.1MC1
(SEQ ID NO: 21).
FIGS. 2A & 2B Identification of protospacer adjacent motif
(PAM) sequences of the Type I-B CRISPR-Cas system in C.
tyrobutyricum. FIG. 2A provides a map of plasmids used in
systematic mutagenesis assays, including the protospacer (SEQ ID
NO: 21) with a 5' PAN sequence. Mutation positions were indicated
on the PAM sequence. Array2-1 (Table 1) was used as the
protospacer. FIG. 2B presents data in a bar graph testing several
variant PAM sequences used in the assay and their corresponding
transformation efficiencies. The plasmid pMTL82151 (PAM, -;
Mutation position, -) was used as the control. Data are based on at
least two independent replicates.
FIGS. 3A-3D: Markerless genome editing in C. tyrobutyricum using
the endogenous Type I-B CRISPR-Cas system. FIG. 3A provides a
schematic drawing that illustrates the steps involved in deleting
the spo0A gene via a lactose inducible CRISPR-Cas system. The
lactose inducible promoter was used to drive the transcription of
synthetic CRISPR array, wherein the array comprises a spacer
(diamonds) separated by 30-nt direct repeats (rectangles). .about.1
kb upstream and downstream homology arms (flanking the native spo0A
gene) were used for the deletion of spo0A gene. Two screening steps
are involved in the process. In the first step, the plasmid was
transformed into C. tyrobutyricum under the selection of
thiamphenicol (Tm). In the second step, lactose was applied to
induce the transcription of synthetic CRISPR array and eliminate
the wild type background cells, thus selecting for the desirable
mutant. Pairs of half arrows and the numbers in the figure indicate
the cPCR target regions and the PCR amplicon sizes, respectively.
FIG. 3B is a table presenting the various plasmids carrying the
CRISPR-Cas9/nCas9/AsCpf1 and Type I-B CRISPR-Cas systems that were
tested for the deletion of spo0A. Promoters and the length of
spacers were optimized for the CRISPR-Cas system in order to
improve the transformation efficiency and editing efficiency. The
inducible promoters tested include the lactose inducible promoter
(Plac) and the arabinose inducible promoter (Para). FIG. 3C
provides data in a bar graph format showing the transformation
efficiency of different plasmids. Data are based on at least two
independent replicates. FIG. 3D provides data in a bar graph format
demonstrating the genome editing efficiency of different plasmids
that can be transformed into C. tyrobutyricum. Fifteen colonies of
each transformant were picked and screened for mutation. The
editing efficiency were calculated as the ratio of the number of
spo0A mutants to the total of fifteen colonies.
FIGS. 4A-4C: Multiplex gene editing in C. tyrobutyricum using the
inducible endogenous Type I-B CRISPR-Cas system. FIG. 4A provides a
schematic drawing illustrating the use of the lactose inducible
CRISPR-Cas system to conduct a double deletion of both the spo0A
and pyrF genes. The deletion vector comprises a CRISPR array under
the control of a lactose promoter and including spacers (diamonds)
targeting the spo0A and pyrF genes, respectively, where each spacer
is flanked by a 30 nucleotide direct repeat (rectangles) and a
nucleic acid sequence of .about.1.2 kb upstream and downstream of
both spo0A and pyrF, respectively (.about.300 bp each) used to
create homology arms to induce homologous recombination after
cleavage by the CRISPR-Cas system. The screening procedure of
double deletion was similar with that for single deletion, except
that a series of subculturing was required before plating the
culture on the TGYLTU plates. Pairs of half arrows and the numbers
in the figure indicate the cPCR target regions and the PCR amplicon
sizes, respectively. Detection of gene deletion events was carried
out at the 8th (FIG. 4B) and 15th (FIG. 4C) generations during the
subculturing. Single deletion vectors pJZ77-Plac-30spo0A and
pJZ77-Plac-30pyrF were used as controls. 47 colonies of each
transformant were picked and screened for mutations. The white
rectangles, grey rectangles, and black rectangles represent wild
type strain, single deletion mutant of spo0A or pyrF, and double
deletion mutant, respectively.
FIG. 5 provides a schematic diagram of the metabolic pathway of
.DELTA.cat1::adhE1 and .DELTA.cat1::adhE2 mutants. The major
products of the two mutants are ethanol and butanol and the
biosynthesis pathways which are absent in the wild type strain are
shown in grey boxes. The butyrate biosynthesis pathway which is
disrupted from the wild type strain is shown with dotted lines. Key
genes in the pathway: pfor, pyruvate::ferredoxin oxidoreductase;
hyda, hydrogenase; fnor, ferredoxin NAD.sup.+ oxidoreductase; pta,
phosphotransacetylase; ack, acetate kinase; thl, thiolase; hbd,
beta-hydroxybutyryl-CoA dehydrogenase; crt, crotonase; bcd,
butyryl-CoA dehydrogenase; cat1, butyrate:acetate coenzyme A
transferase; adhE1/adhE2, aldehyde-alcohol dehydrogenase.
FIGS. 6A & 6B show alignments of the C. tyrobutyricum and C.
pasteurianum leader sequences (FIG. 6A; SEQ ID NO: 23 and 24,
respectively) and the C. tyrobutyricum Array1, Array2 and C.
pasteurianum direct repeat sequences (FIG. 6B; SEQ ID NO: 18, 2 and
25, respectively) of the CRISPR array.
FIGS. 7A-7E: Fermentation profiles of C. tyrobutyricum
WT(pJZ98-Pcat1-adhE1) and mutant .DELTA.cat1::adhE1 strains. Graphs
are provided demonstrating the amount of glucose
(.tangle-solidup.), acetate (.circle-solid.), ethanol
(.largecircle.), butyrate (.DELTA.) and butanol (.box-solid.)
detected over time when C. tyrobutyricum strains are cultured under
different temperatures, using glucose as a carbon source. FIG. 7A
provides the results from culturing WT(pJZ98-Pcat1-adhE1) at
37.degree. C.; FIG. 7B provides the results from culturing mutant
.DELTA.cat1::adhE1 at 37.degree. C.; FIG. 7C provides the results
from culturing mutant .DELTA.cat1::adhE1 at 30.degree. C.; FIG. 7D
provides the results from culturing mutant .DELTA.cat1::adhE1 at
25.degree. C.; and FIG. 7E provides the results from culturing
mutant .DELTA.cat1::adhE1 at 20.degree. C. Values are based on at
least two independent replicates.
FIG. 8A-8E: Fermentation profiles of C. tyrobutyricum
WT(pJZ98-Pcat1-adhE1) and mutant .DELTA.cat1::adhE2 strains. Graphs
are provided demonstrating the amount of glucose
(.tangle-solidup.), acetate (.circle-solid.), ethanol
(.largecircle.), butyrate (.DELTA.) and butanol (.box-solid.)
detected over time when C. tyrobutyricum strains are cultured under
different temperatures, using glucose as a carbon source. FIG. 8A
provides the results from culturing WT(pJZ98-Pcat1-adhE2) at
37.degree. C.; FIG. 8B provides the results from culturing mutant
.DELTA.cat1::adhE2 at 37.degree. C.; FIG. 8C provides the results
from culturing mutant .DELTA.cat1::adhE2 at 30.degree. C.; FIG. 8D
provides the results from culturing mutant .DELTA.cat1::adhE2 at
25.degree. C.; and FIG. 8E provides the results from culturing
mutant .DELTA.cat1::adhE2 at 20.degree. C. Values are based on at
least two independent replicates.
DETAILED DESCRIPTION
Definitions
In describing and claiming the invention, the following terminology
will be used in accordance with the definitions set forth
below.
The term "about" as used herein means greater or lesser than the
value or range of values stated by 10 percent, but is not intended
to designate any value or range of values to only this broader
definition. Each value or range of values preceded by the term
"about" is also intended to encompass the embodiment of the stated
absolute value or range of values.
As used herein an "amino acid modification" defines a substitution,
addition or deletion of one or more amino acids, and includes
substitution with or addition of any of the 20 amino acids commonly
found in human proteins, as well as atypical or non-naturally
occurring amino acids.
The term "substantially purified polypeptide/nucleic acid" refers
to a polypeptide/nucleic acid that may be substantially or
essentially free of components that normally accompany or interact
with the polypeptide/nucleic acid as found in its naturally
occurring environment.
A "recombinant host cell" or "host cell" refers to a cell that
includes an exogenous polynucleotide, regardless of the method used
for insertion. The exogenous polynucleotide may be maintained as a
nonintegrated vector, for example, a plasmid, or alternatively, may
be integrated into the host genome.
As to amino acid sequences, one of ordinary skill in the art will
recognize that individual substitutions, deletions or additions to
a nucleic acid, peptide, polypeptide, or protein sequence which
alters, adds or deletes a single amino acid or a small percentage
of amino acids in the encoded sequence is a "conservatively
modified variant" where the alteration results in the deletion of
an amino acid, addition of an amino acid, or an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are known to those of
ordinary skill in the art. The following eight groups each contain
amino acids that are conservative substitutions for one another: 1)
Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)
Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),
Threonine (T); and 8) Cysteine (C), Methionine (M)
The term "linkage" or "linker" is used herein to refer to groups or
bonds that normally are formed as the result of a chemical reaction
and typically are covalent linkages.
An "operable linkage" is a linkage in which a promoter sequence or
promoter control element is connected to a polynucleotide sequence
(or sequences) in such a way as to place transcription of the
polynucleotide sequence under the influence or control of the
promoter or promoter control element. Two DNA sequences (such as a
polynucleotide to be transcribed and a promoter sequence linked to
the 5' end of the polynucleotide to be transcribed) are said to be
operably linked if induction of promoter function results in the
transcription of an RNA.
The term "isolated" requires that the referenced material be
removed from its original environment (e.g., the natural
environment if it is naturally occurring). For example, a
naturally-occurring polynucleotide present in a living animal is
not isolated, but the same polynucleotide, separated from some or
all of the coexisting materials in the natural system, is
isolated.
As used herein, the term "peptide" encompasses a sequence of 3 or
more amino acids and typically less than 50 amino acids, wherein
the amino acids are naturally occurring or non-naturally occurring
amino acids. Non-naturally occurring amino acids refer to amino
acids that do not naturally occur in vivo but which, nevertheless,
can be incorporated into the peptide structures described
herein.
As used herein, the terms "polypeptide" and "protein" are terms
that are used interchangeably to refer to a polymer of amino acids,
without regard to the length of the polymer. Typically,
polypeptides and proteins have a polymer length that is greater
than that of "peptides."
As used herein a general reference to a polypeptide is intended to
encompass polypeptides that have modified amino and carboxy
termini. For example, an amino acid chain comprising an amide group
in place of the terminal carboxylic acid is intended to be
encompassed by an amino acid sequence designating the standard
amino acids.
As used herein an amino acid "substitution" refers to the
replacement of one amino acid residue by a different amino acid
residue.
As used herein, the term "CRISPR-Cas system" defines a complex
comprising a Cas protein and a spacer RNA.
The terms "target sequence," "target DNA," and "target site" are
used interchangeably to refer to the specific sequence in
chromosomal DNA to which the engineered CRISPR-Cas system is
targeted, and the site at which the engineered CRISPR-Cas system
modifies the DNA.
The terms "upstream" when used in the context of a nucleic acid
sequence, identifies a nucleic acid sequence that is located on the
5' side of a reference nucleic acid sequence. For example a
promoter is located upstream of a nucleic acid coding sequence.
The terms "downstream" when used in the context of a nucleic acid
sequence identify nucleic acid sequence that are located on the 3'
side of a reference nucleic acid sequence. For example a
transcriptional terminator sequence is located downstream of a
nucleic acid coding sequence.
The term "direct repeat sequence" defines an RNA strand that
participates in recruiting a CRISPR endonucleases to the target
site.
As used herein the term "guide sequence" or "spacer" defines a DNA
sequence that transcribes an RNA strand that hybridizes with the
target DNA.
The term "protospacer" refers to the DNA sequence targeted by a
spacer sequence. The protospacer typically comprises the spacer
sequence covalently linked to a protospacer adjacent motif (PAM).
PAM is a 2-6-base pair DNA sequence immediately preceding or
following the DNA sequence targeted by the Cas nuclease in the
CRISPR-Cas system. In some embodiments, the protospacer sequence
hybridizes with the spacer sequence of the CRISPR-Cas system.
The term "endogenous" as used herein, refers to a natural state.
For example a molecule (such as a direct repeat sequence)
endogenous to a cell is a molecule present in the cell as found in
nature. A "native" compound is an endogenous compound that has not
been modified from its natural state.
As used herein, the term "exogenous" refers to a molecule not
present in the composition found in nature. A nucleic acid that is
exogenous to a cell, or a cell's genome, is a nucleic acid that
comprises a sequence that is not native to the cell/cell's
genome.
EMBODIMENTS
As disclosed herein, an efficient genome editing tool for C.
tyrobutyricum, is provided, based on the endogenous Type I-B
CRISPR-Cas system. Advantageously, this novel genome editing tool
has been used to modify the genome of Clostridium strain to produce
a novel strain having improved production of butanol.
In accordance with one embodiment a recombinant microorganism is
provided that produces butanol while the microorganism is cultured
under conditions favorable for growth. In particular, in one
embodiment a microorganism has been modified for increased
expression of aldehyde dehydrogenase activity by the addition of an
exogenous gene that encodes for aldehyde dehydrogenase activity,
optionally wherein the ability of the cat1 gene to produce a
functional protein has been decreased or eliminated. In one
embodiment the recombinant microorganism has been modified by the
integration of an exogenous gene encoding for aldehyde
dehydrogenase activity, optionally wherein the exogenous gene also
encodes for alcohol dehydrogenase activity. In one embodiment the
dehydrogenase activity is an alcohol dehydrogenase activity. In one
embodiment the exogenous gene encodes for both aldehyde
dehydrogenase activity and alcohol dehydrogenase activity. In one
embodiment the exogenous gene is an aldehyde/alcohol dehydrogenase
gene having at least about 80%, 85%, 90%, 95% or 99% sequence
identity to SEQ ID NO: 133 or SEQ ID NO: 134. In one embodiment the
exogenous gene is the adhE1 or adhE2 gene from C.
acetobutylicum.
In one embodiment the modified microorganism is a Clostridium
strain, including for example a Clostridium strain selected from
the group consisting of Clostridium butyricum, Clostridium
thermobutyricum, Clostridium cellulovorans, Clostridium
carboxidivorans, Clostridium tyrobutyricum, Clostridium
polysaccharolyticum, Clostridium populeti, and Clostridium
kluyveri. In one embodiment the Clostridium strain is Clostridium
tyrobutyricum.
In one embodiment a recombinant Clostridium strain modified for
enhanced butanol production is provided wherein the Clostridium
strain comprises an exogenous aldehyde dehydrogenase gene inserted
in to the genome of the Clostridium strain and a modification to
the native cat1 gene, wherein the modification inhibits or prevents
expression of a functional cat1 gene product. In one embodiment the
exogenous aldehyde dehydrogenase gene encodes for both alcohol
dehydrogenase and aldehyde dehydrogenase activity, including for
example a C. acetobutylicum gene selected from the group consisting
of adhE1 and adhE2. In one embodiment the dehydrogenase gene is an
adhE1 gene that encodes a protein having at least 80%, 85%, 90%,
95% or 99% sequence identity to SEQ ID NO: 133. In one embodiment
the dehydrogenase gene is an adhE2 gene that encodes a protein
having at least 80%, 85%, 90%, 95% or 99% sequence identity to SEQ
ID NO: 134. In accordance with one embodiment a modified
Clostridium is provided wherein the cat1 gene is modified by the
insertion of an adhE1 or adhE2 gene into the cat1 gene rendering
the cat1 gene incapable of expressing a functional gene
product.
In accordance with one embodiment a modified strain of Clostridium
is provided wherein butanol is produced by the organism at a level
of at least 15 g/L, when the cells are cultured at a temperature
selected from about 20.degree. C. to about 30.degree. C. in the
presence of a carbon source such as glucose. In accordance with one
embodiment a modified strain of Clostridium is provided wherein
butanol is produced by the organism at a level of at least 20 g/L,
when the cells are cultured at a temperature selected from about
20.degree. C. to about 30.degree. C. In accordance with one
embodiment a modified strain of Clostridium is provided wherein
butanol is produced by the organism at a level of at least 15 g/L
wherein the levels of acetate and ethanol are less than 10 g/L,
when the cells are cultured at a temperature selected from about
20.degree. C. to about 30.degree. C.
In accordance with one embodiment a recombinant Clostridium strain
is provided, wherein the strain when cultured at a temperature of
less than 30.degree. C. using glucose as a carbon source, produces
at least 20 g/L of butanol, and less than 15 g/L of acetate, after
72 hours of culture. In accordance with one embodiment a
recombinant Clostridium strain is provided, wherein the strain when
cultured at a temperature of selected from a range of about
20.degree. C. to about 30.degree. C. using glucose as a carbon
source, produces at least 25 g/L of butanol, and less than 15 g/L
of acetate, after 120 hours of culture. In one embodiment the
Clostridium strain is Clostridium tyrobutyricum.
In one embodiment a Clostridium strain modified for enhanced
butanol production is provided wherein the strain comprises an
exogenous gene encoding for aldehyde dehydrogenase activity, and a
modified native Clostridium cat1 gene, wherein the modification
prevents expression of a functional cat1 gene product, further
wherein the modified strain, when cultured at a temperature of less
than 30.degree. C. using glucose as a carbon source, produces at
least 20 g/L of butanol after 72 hours of culture. In one
embodiment the exogenous gene is inserted into the cat1 gene
rendering the cat1 gene incapable of expressing a functional gene
product. In one embodiment the exogenous gene is an adhE gene
having at least 95% sequence identity to SEQ ID NO: 133 or SEQ ID
NO: 134. In one embodiment the exogenous gene is an adhE1 or adhE2
gene.
In one embodiment a Clostridium strain modified for enhanced
butanol production is provided wherein the strain comprises a
modification to the native cat1 gene, wherein the modification
preventing expression of a functional cat1 gene product, and an
exogenous sequence encoding i) an aldehyde dehydrogenase; ii) a
bifunctional aldehyde/alcohol dehydrogenase; or iii) an aldehyde
dehydrogenase and an alcohol dehydrogenase. In one embodiment the
Clostridium strain is a recombinant organism wherein the cat1 gene
is modified by the insertion of the exogenous sequence into the
cat1 gene rendering the cat1 gene incapable of expressing a
functional gene product. More particularly, in one embodiment the
recombinant Clostridium strain the inserted exogenous sequence
comprises an bifunctional alcohol/aldehyde dehydrogenase gene
selected from the group consisting of adhE1 and adhE2, wherein the
strain, when cultured at a temperature of less than 30.degree. C.
using glucose as a carbon source, produces at least 20 g/L of
butanol after 72 hours of culture.
In accordance with one embodiment a recombinant Clostridium strain
modified for enhanced butanol production is provided wherein the
Clostridium strain comprises an exogenous gene encoding for
aldehyde dehydrogenase activity inserted into the genome of the
strain, and a modified native Clostridium cat1 gene, wherein the
modification to the native Clostridium cat1 gene prevents
expression of a functional cat1 gene product. In one embodiment,
the recombinant Clostridium strain, when cultured at a temperature
of less than 30.degree. C. using glucose as a carbon source,
produces at least 20 g/L of butanol and less than 15 g/L of acetate
after 72 hours of culture. In one embodiment the exogenous gene
encoding for aldehyde dehydrogenase activity is an adhE1 or adhE2
gene that is inserted into the Clostridium native cat1 gene
rendering the cat1 gene incapable of expressing a functional gene
product. In one embodiment a modified Clostridium tyrobutyricum
strain (Clostridium tyrobutyricum JZ100) is provided that has
enhanced production of butanol relative to the native strain. A
representative sample of this modified strain was deposited in
accordance with the provisions of the Budapest Treaty on Nov. 5,
2017, with the Agriculture Research Culture Collection (NRRL), an
International Depository Authority located at 1815 N. University
Street, Peoria, Ill. 61604, and assigned accession number
B-67519.
In accordance with one embodiment the novel modified microorganisms
described herein are used in methods of producing butanol and other
biofuels. In certain of these embodiments, the methods include
culturing one or more different recombinant microorganisms in a
culture medium, and accumulating butanol in the culture medium. In
one embodiment a method of producing butanol is provided wherein a
recombinant Clostridium strain modified for enhanced butanol
production is cultured under conditions suitable for growth of the
strain, and the butanol produced by the cells are recovered. In one
embodiment the cultured Clostridium strain is a strain that has
been modified to inactivate the native cat1 gene, and further
modified to have enhanced aldehyde dehydrogenase and alcohol
dehydrogenase activity. In one embodiment the enhanced aldehyde
dehydrogenase activity is provided by introducing an exogenous
aldehyde dehydrogenase gene into the Clostridium strain, optionally
inserting an exogenous aldehyde dehydrogenase into genome of the
cell and in one embodiment inserting the aldehyde dehydrogenase
gene into the native cat1 gene and thus inactivating the cat1 gene.
In one embodiment the exogenous aldehyde dehydrogenase gene is a
bifunctional aldehyde/alcohol dehydrogenase including for example
adhE1 or adhE2.
In one embodiment the method of producing butanol comprises
culturing a novel Clostridium strain as disclosed herein at a
temperature less than 37.degree. C. Optionally the Clostridium
strain is cultured at a temperature selected from the range of
about 20.degree. C. to about 35.degree. C., or about 20.degree. C.
to about 30.degree. C., or about 25.degree. C. to about 30.degree.
C., or about 20.degree. C. to about 25.degree. C., or at about
30.degree. C., or at about 25.degree. C. or at about 20.degree.
C.
In accordance with one embodiment a method of editing a bacterial
genome is provided that is based on a modified endogenous CRISPR
array. One embodiment of the present disclosure is directed to an
enhanced butanol producing Clostridium strain produced by the novel
CRISPR-CAS system disclosed herein and the use of such novel
strains to produce butanol.
In one embodiment the novel CRISPR-CAS system comprises an
endogenous CRISPR array under the control of an inducible promoter
that drives the expression of a spacer RNA that targets a
protospacer sequence contained within a bacterial genome, resulting
in a double strand break in the targeted DNA. In one embodiment a
method of modifying a Clostridium strain comprises introducing an
exogenous nucleic acid (i.e., a vector) into the bacterial cell
wherein the exogenous nucleic acid comprises a sequence that
encodes a synthetic CRISPR array under the control of an inducible
promoter. In one embodiment the synthetic CRISPR array comprises a
first and second direct repeat, a spacer polylinker site, wherein
the spacer polylinker site is located between the first and second
direct repeat, and a CRISPR terminator sequence located after the
second direct repeat. The spacer polylinker site provides a
plurality of restriction enzyme target sequences that allow for the
easy insertion of a spacer sequence of choice. Advantageously, this
vector allows one to substitute sequences to direct the CRISPR-CAS
system to modify a target protospacer sequence of choice present in
the bacterial genome. The modification of the target sequence can
be enhanced by including sequences that are homologous to the
upstream and/or downstream regions of the target protospacer.
Accordingly, in one embodiment the exogenously introduced nucleic
acid (vector) comprises a homology arm polylinker site, wherein the
homology arm polylinker site comprises a plurality of restriction
enzyme target sequences, that differ from those of the spacer
polylinker site, and allow for the easy insertion of sequences
homologous to the upstream and/or downstream regions of the target
protospacer.
In one embodiment the first and second direct repeat are based on
the endogenous Type I-B CRISPR-Cas system of C. tyrobutyricum. The
direct repeats will typically be identical in sequence relative to
one another but in one embodiment the directs repeat sequences can
vary by one or two nucleotide differences or the two direct repeats
can have greater than 95% or 99% sequence identity to one another
and are orientated relative to each other as direct repeated
sequences on either side of a spacer polylinker/spacer sequence. In
one embodiment the direct repeats comprise a sequence that has at
least 80%, 85%, 90% 95% or 99% sequence identity to SEQ ID NO: 2.
In one embodiment the two direct repeat sequences independently
comprise a sequence having at least 95% sequence identity to the
sequence of SEQ ID NO: 2. In one embodiment the two direct repeat
sequences each comprise the sequence of SEQ ID NO: 2.
In one embodiment the exogenously nucleic acid sequence further
comprises sequence encoding for a Clostridium tyrobutyricum Cas
protein. A vector that further comprises the Clostridium
tyrobutyricum Cas protein can beneficially be used to induce
modifications into Clostridium strains other than Clostridium
tyrobutyricum through the use of the CRISPR-Cas system disclosed
herein.
In accordance with one embodiment a vector for introducing
modifications into a target genomic site of bacteria via a
CRISPR-Cas complex is provided, wherein the target genomic site is
a contiguous nucleic acid sequence comprising a first protospacer
sequence, a first upstream sequence and a first downstream
sequence. More particularly, in one embodiment the vector comprises
a synthetic CRISPR array, an inducible promoter operably linked to
the synthetic CRISPR array and a first homology arm polylinker
site, wherein the synthetic CRISPR array comprises a first and
second direct repeat, a first spacer polylinker site, wherein the
first spacer polylinker site is located between the first and
second direct repeat and a CRISPR terminator sequence located after
the second direct repeat. In one embodiment first and second direct
repeat independently comprise a sequence having at least 95%
sequence identity to the sequence of SEQ ID NO: 2, and the CRISPR
terminator sequence comprises a sequence having at least 95%
sequence identity to the sequence of SEQ ID NO: 3. In one
embodiment the first and second direct repeat each comprise the
sequence of SEQ ID NO: 2, and the CRISPR terminator sequence
comprises the sequence of SEQ ID NO: 3. In one embodiment the
inducible promoter is any bacterial promoter known to those skilled
in the art whose promoter activity can be regulated by one or more
inducer agents. In one embodiment the inducible promoter is a
lactose inducible promoter and the inducing agent is lactose or a
lactose analog such as IPTG. In one embodiment the vector further
comprises a native Clostridium tyrobutyricum Cas encoding sequence,
optionally wherein the native Clostridium tyrobutyricum Cas
encoding sequence is operably linked to an inducible promoter.
The vectors described herein can be further modified for multiplex
editing of multiple target sites based on the number of spacer
sequences are present in the inducible CRISPR array. For example,
in one embodiment a vector is provided for introducing
modifications into a first and second target genomic site of
bacteria via a CRISPR-Cas complex of the present disclosure. In
this embodiment a first target genomic site is a contiguous nucleic
acid sequence comprising a first protospacer sequence, a first
upstream sequence and first downstream sequence, and the second
target genomic site is a contiguous nucleic acid sequence
comprising a second protospacer sequence, a second upstream
sequence and second downstream sequence, and the vector comprises a
first and second homology arm polylinker site. The synthetic CRISPR
array of such a vector comprises a first, second and third direct
repeat, wherein the wherein the first second and third direct
repeat comprises a sequence having at least 95% sequence identity
to the sequence of SEQ ID NO: 2. Optionally the first, second and
third direct repeat sequence are identical to SEQ ID NO: 2. The
synthetic CRISPR array further comprises a first and second spacer
polylinker site, wherein the first spacer polylinker site located
between the first and second direct repeat, and wherein the second
spacer polylinker site located between the second and third direct
repeat, optionally wherein the synthetic CRISPR array further
comprises a CRISPR terminator sequence is located after the third
direct repeat. In one embodiment the CRISPR terminator sequence
comprises the sequence of SEQ ID NO: 3.
In one embodiment the vector comprises a first spacer sequence
inserted into the first spacer polylinker site and a first and
second homology arm sequence inserted into the first homology arm
polylinker site, wherein the first homology arm sequence comprises
a nucleotide sequence sharing at least about 90%, 95% or 99%
sequence identity to the first upstream sequence, and the second
homology arm comprises a nucleotide sequence sharing at least about
90%, 95% or 99% sequence identity to the first downstream sequence.
In one embodiment the spacer sequence is 10 to 100, or 20 to 60, or
20 to 50, or 25 to 50 or 30 to 40 nucleotides in length. In one
embodiment the spacer comprises the sequence of SEQ ID NO: 4. In
one embodiment the first homology arm sequence comprises a
nucleotide sequence having 100% sequence identity to the first
upstream sequence, and the second homology arm comprises a
nucleotide sequence having 100% sequence identity to the first
downstream sequence.
In embodiments targeting two or more target protospacer sequences
in a bacterial genome the vector comprises
a first spacer sequence inserted into the first spacer polylinker
site;
a second spacer sequence of inserted into the second spacer
polylinker site;
a first and second homology arm sequence inserted into the first
homology arm polylinker site, wherein the first homology arm
sequence comprises a nucleotide sequence sharing at least about
90%, 95% or 99% sequence identity to the first upstream sequence,
and the second homology arm comprises a nucleotide sequence sharing
at least about 90%, 95% or 99% sequence identity to the first
downstream sequence; and
a third and fourth homology arm sequence inserted into the second
homology arm polylinker site, wherein the third homology arm
sequence comprises a nucleotide sequence sharing at least about
first homology arm sequence comprises a nucleotide sequence sharing
at least about 90%, 95% or 99% sequence identity to the second
upstream sequence, and the second homology arm comprises a
nucleotide sequence sharing at least about 90%, 95% or 99% sequence
identity to the second downstream sequence.
The present disclosure further encompasses any bacterial strain
comprising an inducible CRISPR array vector of the present
disclosure.
In accordance with one embodiment a method of producing butanol is
provided wherein the method comprises the steps of culturing a
Clostridium strain modified in accordance with the present
disclosure to produce increased levels of butanol relative to the
unmodified strain under conditions suitable for growth of the
strain. In one embodiment the method comprises culturing the strain
in the presence of a carbon source such as glucose or other sugar
at a temperature at or below 37.degree. C. In one embodiment the
cells are cultured at a temperature below 37.degree. C., optionally
at a temperature selected from a range of about 20.degree. C. to
about 35.degree. C.; or about 20.degree. C. to about 30.degree. C.;
or about 25.degree. C. to about 30.degree. C.; or about 30.degree.
C., about 25.degree. C.; or about 20.degree. C. to about 20.degree.
C. The butanol produce by the modified cells can be collected after
48 or 72 hours of culture or longer.
In accordance with one embodiment a method of modifying a target
site of a bacterial cell genome is provided wherein the method
comprises
transforming a bacterial cell with the vector of the present
disclosure and selecting for transformants comprising the
vector;
inducing the expression of the Type I-B CRISPR array; and
identifying recombinant bacteria having a modification to the
target site of the genome. Subsequent to the modification to the
genome, the originally introduced vector can be eliminated from the
cell. In one embodiment the introduced vector exists as an
extra-chromosomal vector that is maintained in the bacterial by a
selectable marker such as an antibiotic resistance gene. In one
embodiment the method comprises targeting the endogenous cat1 gene
and the vector comprises a spacer sequence of
TABLE-US-00001 (SEQ ID NO: 4)
CTTGTAGAAGATGGATCAACCCTACAACTTGGTA.
Example 1
Exploitation of Type I-B CRISPR-Cas of Clostridium tyrobutyricum
for Genome Engineering.
The endogenous Type I-B CRISPR-Cas of Clostridium tyrobutyricum was
analyzed for its ability to function as a tool for modifying
targeted sequence present in the genome of Clostridium
tyrobutyricum. In silico CRISPR array analysis and plasmid
interference assay revealed that TCA or TCG at the 5'-end of the
protospacer was the functional protospacer adjacent motif (PAM) for
CRISPR targeting. With use of a lactose inducible promoter for
CRISPR array expression, applicant significantly decreased the
toxicity of CRISPR-Cas and enhanced the transformation efficiency
of constructs that encoded the CRISPR-Cas complex. Applicants the
effectiveness of the endogenous Type I-B CRISPR-Cas by successfully
deleting the native spo0A gene with an editing efficiency of 100%.
Applicant further evaluated effects of the spacer length on genome
editing efficiency. Interestingly, spacers .ltoreq.20 nt led to
unsuccessful transformation consistently, likely due to severe
off-target effects; while a spacer of 30-38 nt is most appropriate
to ensure successful transformation and high genome editing
efficiency. Moreover, multiplex genome editing for the deletion of
spo0A and pyrF was achieved in a single transformation, with an
editing efficiency of up to 100%. Finally, with the integration of
the aldehyde/alcohol dehydrogenase gene (adhE1 or adhE2) to replace
cat1 (the key gene responsible for butyrate production and
previously could not be deleted), two mutants were created for
n-butanol production, with the butanol titer reached historically
record high of 26.2 g/L in a batch fermentation. Altogether, these
results demonstrate the programmability and high efficiency of
endogenous CRISPR-Cas. The developed protocol herein has a broader
applicability to other prokaryotes containing endogenous CRISPR-Cas
systems. C. tyrobutyricum could be employed as an excellent
platform to be engineered for biofuel and biochemical production
using the CRISPR-Cas based genome engineering toolkit.
Materials and Methods
Bacterial Strains and Cultivation
All the strains used in this study are listed in Table 3. The E.
coli strain NEB Express (New England BioLabs Inc., Ipswich, Mass.)
was used for general plasmid propagation. E. coli CA434 was
employed as the donor strain for conjugation. All E. coli strains
were routinely cultivated in Luria-Bertani (LB) broth or on solid
LB agar plate supplemented with 30 .mu.g/mL chloramphenicol (Cm) or
50 .mu.g/mL kanamycin (Kan) when required. C. tyrobutyricum ATCC
25755 (KCTC 5387) was obtained from the American Type Culture
Collection (ATCC, Manassas, Va., USA) and propagated anaerobically
at 37.degree. C. in Tryptone-Glucose-Yeast extract (TGY) medium. 15
.mu.g/mL thiamphenicol (Tm), 250 g/mL D-cycloserine, 40 mM lactose
or 20 .mu.g/mL uracil was added into the medium when required.
Identification and Analysis of Putative Protospacer Matching CRISPR
Spacers of C. tyrobutyricum
Nucleotide BLAST was used to analyze the CRISPR spacers of C.
tyrobutyricum, by aligning the spacer sequences against the
existing genome sequences in the National Center for Biotechnology
Information (NCBI) database. Putative protospacers were inspected
for their matching with the spacers as the putative invading DNA
elements, such as phage (prophage), plasmid, transposon, integrase,
and so on. For the analysis, we set a maximum of 15% (a maximum of
5/34 mismatching nucleotides) for the mismatches between the
putative protospacer and the corresponding CRISPR spacer of C.
tyrobutyricum.
Plasmid Construction
All the plasmids and primers used in this study are listed in Table
3 and Table 4, respectively. The Phanta Max Super-Fidelity DNA
Polymerase (Vazyme Biotech Co., Ltd., Nanjing, China) was used for
the PCR to amplify DNA fragments for cloning purposes. For the
attempt to delete spo0A gene (CTK_RS09345) in C. tyrobutyricum
using the Type II CRISPR-Cas9 and CRISPR-Cas9 nickase (nCas9)
systems derived from S. pyogenes, the plasmid pYW34-BtgZI was
chosen as the mother vector. This vector contains the Cas9 open
reading frame (ORF) driven by the lactose inducible promoter and
the chimeric gRNA sequence preceded by two BtgZI sites (for easy
re-targeting purpose by inserting the small RNA (sCbei_5830)
promoter along with the 20-nt guiding sequence). The vector
pJZ23-Cas9 was constructed from pYW34-BtgZI through Gibson Assembly
as follows. The erythromycin (Erm) marker and CAK1 replicon of
pYW34-BtgZI were replaced with Cm marker and pBP1 replicon,
respectively, through an in vitro double digestion with Cas9
nuclease following the procedure as described previously (Wang et
al., 2016, ACS Synth. Biol. 5, 721-732). The Cm marker and the pBP1
replicon were amplified from pMTL82151. The TraJ component which is
essential for the conjugation was also amplified from pMTL82151 and
cloned into the ApaI restriction site of pYW34-BtgZI through Gibson
Assembly, generating vector pJZ23-Cas9. To construct pJZ58-nCas9,
the Plac-Cas9 expression cassette within pJZ23-Cas9 was replaced
with the Plac-nCas9 expression cassette as follows. A partial
fragment of the nCas9 ORF which contains the mutation (D10A) was
obtained by PCR using plasmid pMJ841 (Addgene, Cambridge, Mass.,
USA) as the template. Then the partial fragment of nCas9 was fused
with lactose inducible promoter (which was amplified from
pYW34-BtgZI) through Splicing by Overlap Extension (SOE) PCR,
yielding the Plac-nCas9 expression cassette. The Plac-nCas9
expression cassette was cloned into pJZ23-Cas9 by replacing the
Plac-Cas9 fragment between ApaI and NheI restriction sites,
generating pJZ58-nCas9.
Based on pJZ23-Cas9 and pJZ58-nCas9, the small RNA (sCbei_5830)
promoter fused with the 20-nt guiding sequence
(5'-GACATGCTATTGAAGTAGCG-3'; SEQ ID NO: 6) targeting on spo0A and
two homology arms (.about.1 kb each) were cloned into the BtgZI and
NotI sites, respectively, as described previously (Wang et al.,
2017 Appl. Environ. Microbiol. 83, e00233-17), generating
pJZ23-Cas9-spo0A and pJZ58-nCas9-spo0A.
In order to employ the CRISPR-AsCpf1 system derived from
Acidaminococcus sp. BV3L6 to delete spo0A in C. tyrobutyricum, the
plasmid pJZ60-AsCpf1-spo0A was constructed as follows. First,
AsCpf1 was amplified from pDEST-hisMBP-AsCpf1-EC and fused with the
lactose inducible promoter (amplified from pYW34-BtgZI) through SOE
PCR, yielding the Plac-AsCpf1 expression cassette. The Plac-AsCpf1
expression cassette was then cloned into the NdeI restriction site
of pMTL82151 with Gibson Assembly, yielding the plasmid
pWH36-AsCpf1. Based on pWH36-AsCpf1, the small RNA (sCbei_5830)
promoter fused with a synthetic CRISPR-AsCpf1 array and two
homology arms (.about.1 kb each) were cloned into the BamHI site
with Gibson Assembly, generating pJZ60-AsCpf1-spo0A. The synthetic
CRISPR-AsCpf1 array was designed to contain two 20-nt direct repeat
sequences (5'-TAATTTCTACTCTTGTAGAT-3'; SEQ ID NO: 7) separated by
one 23-nt guide sequence (5'-CCGAGAGTAATCGTGCTTTCAGC-3'; SEQ ID NO:
8) targeting on the spo0A gene. The small RNA promoter was used to
drive the expression of the CRISPR-AsCpf1 array (See Wang et al.,
2016).
For the plasmid interference assay, the two primers (see the
`Plasmid interference assays` section in Table 4) for each plasmid
(carrying the protospacer with 5' or 3' PAM) were first annealed,
and then ligated into pMTL82151 which was pre-digested with EcoRI
and BamHI. Plasmid pJZ69-leader-38spo0A was constructed through
Gibson Assembly by cloning a synthetic CRISPR expression cassette
and two homology arms (for spo0A deletion through homologous
recombination) into the vector pMTL82151 between EcoRI and KpnI
sites, and between KpnI and BamHI sites, respectively. The
synthetic CRISPR expression cassette contained a 291 bp native
CRISPR leader sequence, a 38-nt spo0A spacer1 sequence
(5'-ATACCGTTTTCTTGCTCTCACTACTATTAGCTATATCA-3') flanked by two 30-nt
direct repeat sequences (5'-GTTGAACCTTAACATGAGATGTATTTAAAT-3'; SEQ
ID NO: 2) and a 342 bp terminator sequence which was found at the
downstream of the endogenous CRISPR array of C. tyrobutyricum. The
leader sequence, terminator sequence, upstream and downstream
homology arms (.about.1 kb each) of spo0A were obtained by PCR
using the genomic DNA (gDNA) of C. tyrobutyricum as the template
(Table 4). Spacer and direct repeat sequences were included in the
reverse primer for amplifying the leader sequence and the forward
primer for amplifying the terminator. The synthetic CRISPR
expression cassette was obtained by fusing the spacer and direct
repeat sequences through SOE PCR. To construct pJZ74-Plac-38spo0A
and pJZ76-Para-38spo0A, a lactose inducible promoter and an
arabinose inducible promoter were used respectively to replace the
native leader sequence in pJZ69-leader-38spo0A. The lactose
inducible promoter and arabinose inducible promoter were amplified
from the plasmid pYW34-BtgZI and the gDNA of C. acetobutylicum ATCC
824, respectively. Based on pJZ74-Plac-38spo0A, plasmid
pJZ75-Plac-38spo0A was constructed by replacing the 38-nt spo0A
spacer1 sequence with the 38-nt spo0A spacer2 sequence
(5'-GCAACCATAGCTATAAATTCTGAATTTGTTGGTTTACC-3'; SEQ ID NO: 10) which
targeted on another locus of the spo0A gene (Table 4). Plasmids
pJZ74-Plac-10spo0A, pJZ74-Plac-20spo0A, pJZ74-Plac-30spo0A and
pJZ74-Plac-50spo0A (for evaluating spacers of various lengths) were
constructed by replacing the 38-nt spo0A spacer1 sequence in
pJZ74-Plac-38spo0A with the 10-nt spacer1 (5'-ATACCGTTTT-3'; SEQ ID
NO: 11), 20-nt spacer1 (5'-ATACCGTTTTCTTGCTCTCA-3'; SEQ ID NO: 12),
30-nt spacer1 (5'-ATACCGTTTTCTTGCTCTCACTACTATTAG-3'; SEQ ID NO:
13), and 50-nt spacer1
(5'-ATACCGTTTTCTTGCTCTCACTACTATTAGCTATATCATTATTAAACATT-3'; SEQ ID
NO: 14), respectively.
For the double deletion of the spo0A gene and pyrF gene
(CTK_RS12430), the plasmid pJZ77-Plac-30spo0A/30pyrF was
constructed to contain the synthetic CRISPR expression cassette
comprised of the lactose inducible promoter, the native terminator
and a synthetic array sequence carrying two spacer sequences
insulated by three 30-nt direct repeat sequences. The synthetic
CRISPR expression cassette and four homology arms (for deleting the
two genes respectively) were cloned through Gibson Assembly into
pMTL82151 between EcoRI and KpnI sites, and between KpnI and BamHI
sites, respectively. The 30-nt spacer1 targeting on spo0A and the
30-nt spacer3 (5'-TTGGATGTTCTTATAAGGACAAATACTCCT-3'; SEQ ID NO: 15)
targeting on pyrF were used in pJZ77-Plac-30spo0A/30pyrF. The
upstream and downstream homology arms for spo0A deletion
(.about.300 bp each) and for pyrF deletion (.about.300 bp each)
respectively were amplified using the gDNA of C. tyrobutyricum as
template (Table 4). The plasmid pJZ77-Plac-30spo0A (30-nt spacer1,
two arms of .about.300 bp for each) for spo0A single deletion and
the plasmid pJZ77-Plac-30pyrF (30-nt spacer3, two arms of
.about.300 bp for each) for pyrF single deletion were constructed
as the control for the double deletion using the `two-spacer`
approach.
To delete the phosphotransacetylase/acetate kinase operon (pta-ack;
CTK_RS08755-CTK_RS08750), plasmids pJZ86-Plac-34pta/ack was
constructed by replacing the 38-nt spo0A spacer1 sequence in
pJZ74-Plac-38spo0A with the 34-nt pta-ack spacer4
(5'-GATTGTGCTGTAAATCCTGTACCTAATACTGAAC-3'; SEQ ID NO: 16). Upstream
and downstream homology arms (.about.500 bp each; containing
additional KpnI and BamHI recognition sequences in the middle) for
pta-ack operon deletion were amplified using the gDNA of C.
tyrobutyricum as template (Table 4) and cloned into pMTL82151
through Gibson Assembly between KpnI and BamHI sites. The adhE1
gene (CA_P0162) and adhE2 gene (CA_P0035) amplified from the total
DNA of C. acetobutylicum ATCC 824 was inserted into the middle of
the two homology arms of plasmid pJZ86-Plac-34pta/ack between the
additional KpnI and BamHI sites, yielding
pJZ86-Plac-34pta/ack(adhE1) and pJZ86-Plac-34pta/ack(adhE2),
respectively. The constructions of plasmids pJZ95-Plac-34cat1,
pJZ95-Plac-34cat1(adhE1) and pJZ95-Plac-34cat1(adhE2), used for
cat1 gene (CTK_RS03145) deletion or replacement, were similar with
plasmids pJZ86-Plac-34pta/ack, pJZ86-Plac-34pta/ack(adhE1) and
pJZ86-Plac-34pta/ack(adhE2), respectively. The spacer used for
targeting cat1 gene was 34-nt spacer5
(5'-CTTGTAGAAGATGGATCAACCCTACAACTTGGTA-3'; SEQ ID NO: 4). To
construct the plasmid-based adhE1 or adhE2 overexpression vectors,
the promoter of cat1 gene was amplified from the gDNA of C.
tyrobutyricum and cloned into pMTL82151 through Gibson Assembly
between EcoRI and KpnI sites, generating plasmid pJZ98-Pcat1. Then
adhE1 gene and adhE2 gene were cloned into plasmid pJZ98-Pcat1
through Gibson Assembly between BtgZI and EcoRI sites, yielding
pJZ98-Pcat1-adhE1 and pJZ98-Pcat1-adhE2, respectively.
Transformation of C. tyrobutyricum
Plasmids used in this study were transformed into C. tyrobutyricum
via conjugation following published protocols with modifications
(Yu et al., 2012 Appl. Microbiol. Biotechnol. 93, 881-889). The
donor strain E. coli CA434 carrying the recombinant plasmid was
cultivated in LB medium supplemented with 30 .mu.g/mL Cm and 50
.mu.g/mL Kan. When the OD.sub.600 reached 1.5-2.0, about 3 mL E.
coli CA434 cells were centrifuged and washed twice (with 1 mL fresh
LB medium for each wash) to remove the antibiotics. The obtained
donor cells were then mixed with 0.4 mL of the recipient culture of
C. tyrobutyricum (which had an OD.sub.600 of 2.0-3.0 after an
overnight growth in TGY medium). The cell mixture was spotted onto
a well-dried TGY agar plate and incubated in the anaerobic chamber
at 37.degree. C. for mating purposes. After 24 hours, the
transconjugants were collected by washing them off the conjugation
plate using one mL of TGY medium, and were then spread onto TGY
plates containing 15 g/mL Tm and 250 .mu.g/mL D-cycloserine (for
eliminating the residual E. coli CA434 donor cells). Transformant
colonies could be generally observed after 48-96 h of
incubation.
Mutant Screening
The screening of mutants was performed following the protocol as
described previously with modifications (see Wang et al., 2017).
The transformant colonies of C. tyrobutyricum were picked and
inoculated into TGY liquid medium with addition of 15 g/mL Tm
(TGYT). The obtained cultures were then diluted serially and spread
onto TGY plates supplemented with 40 mM lactose and 15 .mu.g/mL Tm
(TGYLT). The plates were incubated anaerobically at 37.degree. C.
until colonies were observed. Colony PCR (cPCR) was then performed
to screen the putative mutants. When the deletion of pyrF is
involved, 20 .mu.g/mL uracil was added into TGYLT medium (TGYLTU)
to support the growth of .DELTA.pyrF strain. When shorter spacer
sequence (30 bp) and shorter homology arms (.about.300 bp) were
used for the gene deletion, a series of subculturing (1% v/v
inoculum) was carried out using either TGYLT or TGYLTU liquid
medium to enrich the desirable homologous recombination, before
plating the culture onto the TGYLT or TGYLTU plates for
selection.
Batch Fermentation
Batch fermentations with various C. tyrobutyricum strains were
carried out in 500 mL bioreactors (GS-MFC, Shanghai Gu Xin
biological technology Co., Shanghai, China) with a 250 mL working
volume. The fermentation medium used in this study was prepared as
described previously (Zhang et al., 2017, Biotechnol. Bioeng. 114,
1428-1437), which comprised (per liter of distilled water): 110 g
glucose; 5 g yeast extract; 5 g tryptone; 3 g
(NH.sub.4).sub.2SO.sub.4; 1.5 g K.sub.2HPO.sub.4; 0.6 g
MgSO.sub.4.7H.sub.2O; 0.03 g FeSO.sub.4.7H.sub.2O, and 1 g
L-cysteine. The C. tyrobutyricum strain was first incubated
anaerobically at 37.degree. C. in TGY medium until OD.sub.600
reached 1.5 and then the active seed culture was inoculated into
the bioreactor at a volume ratio of 5%. The fermentation was
carried out at pH 6.0 under various temperatures (20, 25, 30,
37.degree. C.). Batch fermentations with C. beijerinckii NCIMB 8052
and C. saccharoperbutylacetonicum N1-4 under various temperatures
(20, 25, 30, 35.degree. C.) were carried out as described
previously. Samples were taken every 12 hours for the analysis.
Analytical Methods
Cell growth was determined by measuring the optical density at 600
nm (OD.sub.600) using a cell density meter (Ultrospec 10, Biochrom
Ltd., Cambridge, England). Glucose, acetate, ethanol, butyrate and
butanol concentrations in the fermentation broth were analyzed
using an HPLC (Agilent 1260 series, Agilent Technologies, Santa
Clara, Calif., USA) equipped with a refractive index detector (RID)
and an Aminex HPX-87H column (Bio-Rad, Hercules, Calif., USA). 5 mM
H.sub.2SO.sub.4 was used as the mobile phase a flow rate of 0.6
mL/min at 25.degree. C.
Results
Attempts of Genome Editing in C. tyrobutyricum with
CRISPR-Cas9/Cpf1 Systems
Recently, genome editing tools have been developed for several
Gram-positive bacteria based on the Type II CRISPR-Cas9/nCas9
system derived from S. pyogenes, and various Type V CRISPR-Cpf1
systems. These systems were first considered by applicants for
genome engineering in C. tyrobutyricum. The spo0A gene which is the
master regulator for sporulation was selected as the target gene to
delete. To abate the strong toxicity of the nuclease/nickase, we
constructed CRISPR-Cas9/nCas9/AsCpf1 based vectors by placing the
Cas9/nCas9/AsCpf1 encoding gene under the control of a lactose
inducible promoter, whereas the gRNA/crRNA were expressed from the
constitutive small RNA promoter from C. beijerinckii. (Wang et al.,
2016) In addition, the homology arms for spo0A deletion through
homologous recombination were inserted into the same plasmid (Wang
et al., 2016). The resultant plasmid (pJZ23-Cas9-spo0A,
pJZ58-nCas9-spo0A and pJZ60-AsCpf1-spo0A, respectively; FIG. 3A)
was attempted to be transformed into C. tyrobutyricum. Although
numerous attempts were implemented, no transformant could be
obtained (FIG. 3C). This indicated that, due to the high toxicity
of the heterologous nuclease/nickase and the limited transformation
efficiency of C. tyrobutyricum, the genome editing is difficult to
be realized in this microorganism with the CRISPR-Cas9/nCas9/Cpf1
system. It has been reported that the endogenous CRISPR-Cas system
within bacteria and archaea can be harnessed for genome editing for
the host microorganism (Li et al., 2015 Nucleic Acids Res. 44,
e34). From the genome sequence, we noticed that C. tyrobutyricum
possesses a Type I-B CRISPR-Cas system. Therefore, we next turned
to exploit this endogenous CRISPR-Cas system for genome editing in
C. tyrobutyricum.
In Silico Analysis of the Type I-B CRISPR-Cas System of C.
tyrobutyricum
Based on the genome sequence, two CRISPR arrays were identified
located at two different loci within the C. tyrobutyricum genome.
The first CRISPR array (Array1) contains 17 spacers (length: 34-38
nt) flanked by direct repeat sequences of 30 nt
(5'-ATTGAACCTTAACATGAGATGTATTTAAAT-3'; SEQ ID NO: 18). However, no
putative Cas-encoding gene was found at the upstream or downstream
of Array1. The second CRISPR array (Array2) was comprised of eight
spacers (length: 34-38 nt) flanked by direct repeat sequences of 30
nt (5'-GTTGAACCTTAACATGAGATGTATTTAAAT-3'; SEQ ID NO: 2) which is
only one nucleotide different from that of Array1). A core cas gene
operon (cas6-cas8b-cas7-cas5-cas3-cas4-cas1-cas2) was found at the
upstream of Array2, indicating that this CRISPR-Cas system belongs
to the Type I-B subtype (FIG. 1A).
The CRISPR-Cas system is known as an immune system, and its spacer
sequences are typically derived from the invading genetic elements
during the `adaptation` stage. Therefore, we set out to analyze all
the 25 spacer sequences specified in Array1 and Array2 using
Nucleotide BLAST, aiming to elucidate whether any spacer sequence
matches the putative invading DNA elements, including phage
(prophage), plasmid, transposon, and integrase. In order to
determine the putative protospacers, a mismatch of less than 15% (
5/34 mismatching nucleotides or less) was defined (Shariat et al.,
2015). Among all the 25 spacers in the CRISPR-Cas system of C.
tyrobutyricum, only one spacer sequence (the 17.sup.th spacer
within Array 1, Array1-17:
5'-TGGTATCACCAACTTTTGTCCAGGATATATGAGGTT-3'; SEQ ID NO: 19) hit
(with five mismatches) the putative protospacers found in phage
sequence from C. thermocellum and prophage sequence from
Geobacillus thermoglucosidasius (FIG. 1B).
Identification of Protospacer Adjacent Motif (PAM) Sequences
A plasmid transformation interference assay was carried out to test
the activity of the Type I-B CRISPR-Cas system of C. tyrobutyricum
and meanwhile identify the putative PAM sequences. The plasmid
employed in interference assay contains a protospacer for the DNA
targeting purpose and a 5-nt putative PAM sequence located at the
5'- or 3'-end of the protospacer which is essential for the
recognition by the Type I CRISPR-Cas system (Table 1). Though the
spacer Array1-17 was the only spacer found to match the invading
DNA elements, there was no adjacent Cas-encoding genes associated
with Array1 discovered. Therefore, additionally we decided to
employ another spacer (Array2-1:
GCATTCAGACTTGCAACTGTAACTCCCTAGTACTCCCC; SEQ ID NO: 21) derived from
Array2 as the protospacer for the plasmid interference purpose. The
5-nt sequences derived from the upstream or downstream of
identified putative protospacers were tested as putative PAM
sequences (FIG. 1B & Table 1). Our in silico analysis revealed
that the C. tyrobutyricum CRISPR array possessed high homology to
the CRISPR array of C. pasteurianum, for both the leader and direct
repeat sequences (FIGS. 6A & 6B). Therefore, we hypothesized
that the CRISPR-Cas system of C. tyrobutyricum may share the same
or similar PAM with that of C. pasteurianum. Hence, the PAM
sequences for C. pasteurianum Type I-B CRISPR-Cas system were also
employed in the plasmid interference assay (Table 1). Altogether,
14 interference plasmids were constructed by combining different
protospacer and PAMs (Table 1). Since both a protospacer and PAM
sequence have been included on the interference plasmid, there
would be no transformants (the specific plasmid is cleaved and
eliminated; we define this as the `interference response`) if the
CRISPR-Cas system is functional with a particular combination of
the protospacer and PAM. As shown in Table 1, no matter what PAM
sequences were employed, there was no interference response
observed when the protospacer Array1-17 was used. This result
suggests that Array1 which does not have an adjacent cas gene
operon may be silent in the genome of C. tyrobutyricum, or it was
possibly derived from a gene transfer event which was unrelated to
the development of the CRISPR-Cas immunity system in C.
tyrobutyricum. While combinations of protospacer Array2-1 with 5'
adjacent PAM sequences 5'-CATCA-3' or 5'-TTTCA-3', derived from C.
tyrobutyricum and C. pasteurianum respectively, successfully
triggered the interference response (Table 1). Plasmids contained
combinations of Array2-1 (as the protospacer) and other PAMs were
transformed efficiently into C. tyrobutyricum (Table 1). These
results indicated that Array2 along with the associated core cas
gene operon in C. tyrobutyricum is active and highly functional.
Furthermore, the specific PAM sequence located at the 5'-end of the
protospacer is essential for the target recognition of Cas
proteins.
We used 5-nt PAM sequences in the plasmid transformation
interference assay on the basis that most identified PAMs within
various microorganisms vary between 2-5 nt (Shah et al., 2013).
However, it is noteworthy that the two functional PAM sequences
contain a conserved 3-nt sequence 5'-TCA-3' which may play the
critical role for the target recognition for C. tyrobutyricum Type
I-B CRISPR-Cas system. To test our hypothesis, various PAMs
(5'-NTCA-3' with point mutations at different positions) built upon
5'-TCA-3' were systematically evaluated for their functionality
(FIG. 2). As shown in FIG. 2B, significant differences in the
transformation efficiency were observed with plasmids containing
different PAMs (along with Array2-1 as the protospacer). All the
plasmids contained point mutations at position -4 triggered the
interference response, suggesting that the first three nucleotides
(5'-TCA-3') encompass the core PAM sequence. When `T` located at
position -3 was mutated, only slightly increased transformation
efficiency was obtained, indicating that the nucleotide on position
-3 had a minor effect on target recognition. Nevertheless, high
transformation efficiency (comparable to the control plasmid
pMTL82151) was observed when `C` located at position -2 was mutated
to `G` or `A` or `A` located at position -1 was mutated to `T`. The
transformation efficiency was slightly increased (compared to that
with 5'-NTCA-3') when `A` located at position -1 was mutated to
`C`, while `TCG` kept the similar level of transformation
efficiency with 5'-NTCA-3'. These data demonstrated that, for the
appropriate function of the PAM sequence, pyrimidine nucleotides
(`C` and `T`), rather than purine nucleotides (`G` and `A`), are
more preferable at the position -2, and conversely, purine
nucleotides are better options than pyrimidine nucleotides at the
position -1. Overall, 3-nt sequences 5'-TCA-3' (TCA) and 5'-TCG-3'
(TCG) (also written as TCR collectively for both) which led to an
approximately 1,000-fold drop in plasmid transformation efficiency
(compared to the control plasmid pMTL82151, FIG. 2B) were concluded
to be the functional PAM sequences of the Type I-B CRISPR-Cas
system in C. tyrobutyricum.
TABLE-US-00002 TABLE 1 Effect of different combinations of
protospacers and PAM sequences on the transformation efficiency.
Transform efficiency (.times.10.sup.2 CFU/mL Plasmid 5' PAM
Protospacer.sup.a 3' PAM donor).sup.b pMTL82151 4.9 .+-. 0.6 pIF-1
CATCT TGGTATCACCAACTTTTGTCCAGGATATATGAGGTT (SEQ ID NO: 19) 4.2 .+-.
0.8 pIF-2 CATCA TGGTATCACCAACTTTTGTCCAGGATATATGAGGTT (SEQ ID NO:
19) 3.7 .+-. 0.4 pIF-3 TGGTATCACCAACTTTTGTCCAGGATATATGAGGTT (SEQ ID
NO: 19) AGGAT 4.8 .+-. 0.1 pIF-4
TGGTATCACCAACTTTTGTCCAGGATATATGAGGTT (SEQ ID NO: 19) CGGAT 4.2 .+-.
0.7 pIF-5 AATTG TGGTATCACCAACTTTTGTCCAGGATATATGAGGTT (SEQ ID NO:
19) 3.9 .+-. 0.5 pIF-6 TTTCA TGGTATCACCAACTTTTGTCCAGGATATATGAGGTT
(SEQ ID NO: 19) 3.3 .+-. 0.4 pIF-7 TATCT
TGGTATCACCAACTTTTGTCCAGGATATATGAGGTT (SEQ ID NO: 19) 5.1 .+-. 0.2
pIF-8 CATCT GCATTCAGACTTGCAACTGTAACTCCCTAGTACTCCCC (SEQ ID NO: 22)
3.8 .+-. 0.3 pIF-9 CATCA GCATTCAGACTTGCAACTGTAACTCCCTAGTACTCCCC
(SEQ ID NO: 22) 0 .+-. 0 pIF-10
GCATTCAGACTTGCAACTGTAACTCCCTAGTACTCCCC (SEQ ID NO: 22) AGGAT 3.5
.+-. 0.9 pIF-11 GCATTCAGACTTGCAACTGTAACTCCCTAGTACTCCCC (SEQ ID NO:
22) CGGAT 4.1 .+-. 0.1 pIF-12 AATTG
GCATTCAGACTTGCAACTGTAACTCCCTAGTACTCCCC (SEQ ID NO: 22) 4.0 .+-. 0.7
pIF-13 TTTCA GCATTCAGACTTGCAACTGTAACTCCCTAGTACTCCCC (SEQ ID NO: 22)
0 .+-. 0 .sup.aTwo kinds of protospacers were used here: Array1-17
and Array2-1). .sup.bValues are average .+-. standard deviation
based on at least two independent replicates.
Development of an Inducible CRISPR-Cas System for Genome Editing in
C. tyrobutyricum
After establishing that the endogenous Type I-B CRISPR-Cas system
of C. tyrobutyricum was functional and had high interference
activity against plasmids possessing proper protospacer and PAM
sequences, we then attempted to engineer this system to be a genome
editing tool for C. tyrobutyricum. Two parts are required for such
a system: 1) a synthetic CRISPR expression cassette, containing a
spacer targeting on the specific genome sequence; 2) gene editing
cassette, comprised of a pair of homology arms to achieve
homologous recombination (FIG. 3A). The spo0A gene was selected as
the first target gene to be deleted. The 816 bp spo0A ORF contains
a total of 28 potential PAMs (TCR) including 24 TCA and 4 TCG. One
of the PAM (TCA) along with its downstream protospacer sequence
(38-nt spo0A spacer1) was selected as the target site. Plasmid
pJZ69-leader-38spo0A, comprised of a synthetic CRISPR expression
cassette and a spo0A editing cassette (upstream and downstream
homology arms, .about.1 kb each), was constructed to delete the
spo0A gene in C. tyrobutyricum. In the synthetic CRISPR expression
cassette (SEQ ID NO: 4) comprising the native CRISPR leader (SEQ ID
NO: 1) and terminator sequences (SEQ ID NO: 3) were used to drive
the transcription of synthetic CRISPR array which contained the
38-nt spo0A spacer1 (SEQ ID NO: 9) (flanked by 30-nt direct repeat
sequences (SEQ ID NO: 2). Conjugation was carried out. However, no
transformants were obtained with pJZ69-leader-38spo0A, although the
expected transformation efficiency was obtained with pMTL82151 as
the control. Many attempts have been conducted, and the results
were consistently the same (data not shown).
Therefore, even with the endogenous CRISPR-Cas system, the instant
expression could be highly toxic to the cells and thus no
transformants could be obtained. Generally, the leader sequence of
the CRISPR array contains a promoter for CRISPR array transcription
and a regulatory signal for the uptake of new spacer-repeat
elements. In this study, however, for the genome editing purposes,
only the promoter function of the leader sequence is needed. In
order to reduce the toxicity of endogenous CRISPR-Cas system, a
lactose inducible promoter and an arabinose inducible promoter were
evaluated for the transcription of the synthetic CRISPR array in
place of the native leader sequence (FIGS. 3A & B). The
resultant plasmids pJZ74-Plac-38spo0A and pJZ76-Para-38spo0A were
transformed into C. tyrobutyricum. Transformants were generated
with pJZ74-Plac-38spo0A, with an overall transformation efficiency
of 1.7 CFU/mL donor (FIG. 3C); while the transformation with
pJZ76-Para-38spo0A failed, suggesting that the arabinose inducible
promoter was less stringent than the lactose inducible promoter for
the expression of the CRISPR array in C. tyrobutyricum (FIG. 3C).
As a control (or as a means to further confirm the appropriate PAM
sequence), a 38-nt spo0A spacer2 (corresponding PAM: TCT) was
employed to replace the 38-nt spo0A spacer1 in pJZ74-Plac-38spo0A,
generating plasmid pJZ75-Plac-38spo0A. Results demonstrated that
the transformation efficiency with pJZ75-Plac-38spo0A (.about.18.2
CFU/mL donor) increased more than an order of magnitude compared to
that with pJZ74-Plac-38spo0A (FIG. 3C). The obtained transformants
(with either pJZ74-Plac-38spo0A or pJZ75-Plac-38spo0A) were
cultivated in TGYT medium, and then spread onto TGYLT plates to
induce the expression of the synthetic CRISPR array. Colony PCR was
carried out with randomly picked colonies to screen the spo0A
deletion mutants. Results showed that one out of fifteen (6.7%) of
the tested colonies was spo0A deletion mutant (.DELTA.spo0A) from
the transformants with pJZ75-Plac-38spo0A (FIG. 3D). While all
tested colonies were .DELTA.spo0A mutants from the transformants
with pJZ74-Plac-38spo0A, representing an editing efficiency of 100%
(FIG. 3D). These results confirmed our above conclusion concerning
the PAM sequence: the targeting efficiency of TCA is much higher
than TCT. The .DELTA.spo0A mutant was further verified by Sanger
sequencing (data not shown). Collectively, we proved that with the
inducible endogenous CRISPR-Cas system, efficient genome editing
could be achieved in C. tyrobutyricum.
Effects of Spacer Length on Transformation Efficiency and Genome
Editing Efficiency
In the C. tyrobutyricum genome, a total of 25 spacer sequences were
identified in Array1 and Array2 with lengths ranging from 34 to 38
nt. In order to mimic the feature of the native Type I-B CRISPR
array, the 38-nt spo0A spacer1 was employed to develop the genome
editing platform for the deletion of spo0A. However, it is
reasonable to question whether the length of the spacer has an
effect on the transformation efficiency and genome editing
efficiency of the CRISPR-Cas genome engineering platform. To answer
this question, we replaced the 38-nt spo0A spacer1 in plasmid
pJZ74-Plac-38spo0A with 10 nt, 20 nt, 30 nt, and 50 nt of spo0A
spacer1 (while the PAM sequence TCA was kept the same), yielding
pJZ74-Plac-10spo0A, pJZ74-Plac-20spo0A, pJZ74-Plac-30spo0A, and
pJZ74-Plac-50spo0A, respectively (FIG. 3B). Surprisingly, no
transformant was obtained after several attempts with
pJZ74-Plac-10spo0A or pJZ74-Plac-20spo0A. This might be because the
shorter spacer sequences (<20 nt) led to severe off-target
effects which killed the host cells (FIG. 3C). However, when 30-nt,
38-nt and 50-nt spacers were used, transformation efficiencies of
103.0 CFU/mL donor, 1.7 CFU/mL donor and 0.2 CFU/mL donor were
obtained, respectively (FIG. 3C). The longer spacers can bind more
tightly to the target and thus increase the self-targeting activity
of the endogenous CRISPR-Cas system, which may contribute to the
decreased transformation efficiency. The genome editing efficiency
was also assessed for the transformants obtained with
pJZ74-Plac-30spo0A, pJZ74-Plac-38spo0A or pJZ74-Plac-50spo0A.
Interestingly, colonies of various sizes were observed for the
transformants harboring pJZ74-Plac-30spo0A on the TGYLT plates,
while the colonies from the other two transformants appeared
homogeneous in sizes. Large and small colonies of the transformant
harboring pJZ74-Plac-30spo0A were picked separately to screen for
the .DELTA.spo0A mutant, and editing efficiencies of 93.3% and
13.3% were obtained, respectively. The different genome editing
efficiency for large and small colonies might be due to the low
self-targeting activity of the endogenous CRISPR-Cas system when
30-nt spacer was employed. In this case, some of the host cells
could survive from the selection of the endogenous CRISPR-Cas
system, but their growth was still inhibited. Most of the observed
small colonies were wild type cells with growth inhibited, whereas
most of the large colonies were mutant cells without growth
interference because their target site for the CRISPR-Cas system
had been eliminated. On the other hand, the editing efficiencies of
transformants obtained with pJZ74-Plac-38spo0A or
pJZ74-Plac-50spo0A were both 100% (FIG. 3D).
Multiplex Genome Engineering
As described above, single gene deletion was achieved with high
efficiency using the inducible endogenous CRISPR-Cas system. Here,
we further explored this system for multiplex genome editing in C.
tyrobutyricum. The pyrF gene encoding the enzyme orotidine
5-phosphate decarboxylase (involved in the de novo pyrimidine
biosynthesis) together with the spo0A gene were selected as targets
to delete. In order to have the CRISPR-Cas system target onto two
loci at the same time, we inserted two spacers targeting on spo0A
and pyrF respectively into the same CRISPR array insulated by three
direct repeats (FIG. 4A). Considering that the longer spacer is
more toxic to the host cells as we demonstrated above, 30 nt was
used for both spacers (spo0A spacer1 and pyrF spacer3). In
addition, as we noticed that, with the increase of the plasmid
size, the transformation efficiency decreases dramatically
(especially when the vector size >10 kb; data not shown), we
used shorter homology arms for the deletion of both genes (two
homology arms for the deletion of each gene, with the length of
each arm is .about.300 bp), to keep the final vector size <9 kb
(FIG. 4A). Control plasmids pJZ77-Plac-30spo0A and
pJZ77-Plac-30pyrF were also constructed for deleting spo0A and pyrF
individually by using the same corresponding modules (spacer and
homology arms) in pJZ77-Plac-30spo0A/30pyrF for deleting spo0A and
pyrF, respectively. The three plasmids were successfully
transformed into C. tyrobutyricum, and the resulting transformants
were then spread onto TGYLTU plates. However, no mutant was
detected (47 colonies from each transformant were screened with
cPCR) for any of the three transformants, which was not surprising
considering the reduced editing efficiency when shorter spacers and
homology arms were used. In order to enrich the desirable
homologous recombination, a series of subculturing was performed in
TGYLTU liquid medium. Then mutant screening was performed with cPCR
for the 8.sup.th and 15.sup.th generations of the subculture. For
the 8.sup.th generation, for spo0A and pyrF deletion respectively,
editing efficiencies of 59.6% and 40.4% were obtained with the
one-spacer approach (using pJZ77-Plac-30spo0A and
pJZ77-Plac-30pyrF, respectively), while editing efficiencies of
53.2% and 31.9% were obtained with the two-spacer approach (using
pJZ77-Plac-30spo0A/30pyrF) (FIG. 4B). In addition, double deletion
was also detected with the two-spacer approach, but at a much lower
rate (6.4%) (FIG. 4B). For the 15.sup.th generation, up to 100%
editing efficiencies were observed for spo0A and pyrF deletion with
both one-spacer and two-spacer approaches, which meant that as high
as 100% editing efficiency for the double deletion was also
achieved with the two-spacer approach (FIG. 4C).
Engineered C. tyrobutyricum for Butanol Production
C. tyrobutyricum is a hyper-butyrate producer, indicating that the
metabolic pathway from glucose to butyryl-CoA is highly favorable
(FIG. 5). Therefore, using the high efficient endogenous CRISPR-Cas
system, we attempted to engineer the C. tyrobutyricum for
hyper-butanol production. Two aldehyde/alcohol dehydrogenase genes
(adhE1 and adhE2) which can convert butyryl-CoA to butanol were
chosen to introduce into C. tyrobutyricum. In order to drive more
metabolic flux towards C4 products, the pta-ack operon which was
responsible for acetate formation was initially selected to be
deleted or replaced by adhE1/adhE2 (FIG. 5). However, none of the
attempts was successful (data not shown), suggesting that the
pta-ack operon was vital for C. tyrobutyricum metabolism and thus
cannot be deleted.
In C. tyrobutyricum, cat1 is the essential gene for butyrate
biosynthesis, and the ptb-buk operon as seen in solventogenic
clostridial strains does not exist (FIG. 5). Therefore, we
hypothesized that deletion of cat1 could eliminate butyrate
production, and thus the introduction of adhE1/adhE2 can lead to
the conversion of butyryl-CoA for enhanced butanol production.
However, it was previously reported that the disruption of cat1 was
not achievable (with the mobile group II intron), because the
inactivation of cat1 could likely lead to the inability of the
strain for NADH oxidization (Lee et al., 2016a, mBio 7, e00743-16).
Here, based on the high efficient CRISPR-Cas system for genome
engineering, we attempted to delete the cat1 gene or replace it
with adhE1 or adhE2. Similar as the previous report, the deletion
of cat1 was fruitless despite numerous attempts, however the
replacement of cat1 with adhE1 or adhE2 was successful, yielding
mutants .DELTA.cat1::adhE1 and .DELTA.cat1::adhE2, respectively. As
the control, the recombinants WT(pJZ98-Pcat1-adhE1) and
WT(pJZ98-Pcat1-adhE2) were also obtained by introducing the
plasmid-based adhE1 and adhE2 (driven by the cat1 promoter)
overexpression vectors into C. tyrobutyricum. Initial batch
fermentations were carried out at 37.degree. C. (the optimum
temperature for the cell growth of C. tyrobutyricum). Results
demonstrated that acetate (14.8 g/L), ethanol (9.7 g/L) and butanol
(8.7 g/L) were the major products with a low level of butyrate (1.3
g/L) produced for the control strain WT(pJZ98-Pcat1-adhE1) (Table 2
and FIG. 7A). However, for WT(pJZ98-Pcat1-adhE2), acetate (6.9
g/L), ethanol (7.4 g/L) and butyrate (34.1 g/L) were the major
products, with only a small amount of butanol (2.0 g/L) was
produced (Table 2 and FIG. 8A). As we expected, with the butyrate
biosynthesis pathway replaced with the butanol producing pathway,
mutants .DELTA.cat1::adhE1 and .DELTA.cat1::adhE2 produced
negligible butyrate (0.6-0.8 g/L) but high levels of butanol (15.0
g/L). In addition, significant amounts of acetate (15.1-20.8 g/L)
and ethanol (5.2-5.3 g/L) were also produced by the two mutants
(Table 2 and FIGS. 7B & 8B).
Enhance Butanol Production by Carrying Out Fermentation at Low
Temperatures
It is well known that the limited butanol tolerance of the host is
a major bottleneck for butanol production in microorganisms. Recent
studies showed that lower temperature could alleviate the alcohol
toxicity and thus increase the alcohol production. Therefore, batch
fermentations were further carried out at 30, 25 and 20.degree. C.
with .DELTA.cat1::adhE1 and .DELTA.cat1::adhE2, respectively. As
seen in Table 2 and FIGS. 7B-7E & 8B-8E, the acetate production
was kept at the similar levels at different temperatures. However,
the production of ethanol and butanol was significantly increased
at these lower temperatures. Butanol titers for .DELTA.cat1::adhE1
and .DELTA.cat1::adhE2 obtained at 20.degree. C. were 21.4 and 26.2
g/L, respectively, which increased by 42.7% and 74.7%,
respectively, compared with that obtained at 37.degree. C. While
the total BE (butanol and ethanol) production of .DELTA.cat1::adhE1
and .DELTA.cat1::adhE2 reached the maximum of 35.6 and 38.2 g/L,
respectively at 25.degree. C.
TABLE-US-00003 TABLE 2 Summary of fermentation results for C.
tyrobutyricum mutants at various temperatures.sup.a. Glucose
Temperature consumption Acetate Butyrate Ethanol Butanol Total BE
BE yield Strain (.degree. C.) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L)
(g/g of glucose) WT(pJZ98-Pcat1-adhE1) 37 79.1 14.8 1.3 9.7 8.7
18.4 0.23 WT(pJZ98-Pcat1-adhE2) 37 109.0 6.9 34.1 7.4 2.0 9.4 0.09
.DELTA.cat1::adhE1 37 87.1 20.8 0.6 5.3 15.0 20.3 0.23
.DELTA.cat1::adhE2 37 75.5 15.1 0.8 5.2 15.0 20.2 0.27
.DELTA.cat1::adhE1 30 109.6 22.5 0.8 14.3 17.2 31.5 0.29
.DELTA.cat1::adhE2 30 96.7 12.3 1.3 10.8 21.1 31.9 0.33
.DELTA.cat1::adhE1 25 111.9 22.8 1.3 16.6 19.0 35.6 0.32
.DELTA.cat1::adhE2 25 109.4 13.9 1.8 12.8 25.4 38.2 0.35
.DELTA.cat1::adhE1 20 111.9 21.8 1.6 10.4 21.4 31.8 0.28
.DELTA.cat1::adhE2 20 112.2 15.2 2.4 8.9 26.2 35.1 0.31 .sup.aThe
fermentation profiles are provided in FIGS. 7A-7E & 8A-8E;
values are based on at least two independent replicates.
TABLE-US-00004 TABLE 3 Bacterial strains and plasmids used in
Example 1 Strains/Plasmids Relevant characteristic Sources Strains
E. coli NEB Express fhuA2 [Ion] ompT gal sulA11
R(mcr-73::miniTn10-- New Tet.sup.S)2 [dcm]
R(zgb-210::Tn10--Tet.sup.S) endA1 England
.DELTA.(mcrC-mrr)114::IS10 BioLabs CA434 hsd20(r.sup.B-, m.sup.B-),
recA13, rpsL20, leu, proA2, with (Williams IncPb conjugative
plasmid R702 et al., 1990) C. tyrobutyricum ATCC 25755 KCTC 5387,
wild type stain ATCC .DELTA.spo0A Derived from ATCC 25755, with
spo0A gene This work deleted .DELTA.pyrF Derived from ATCC 25755,
with pyrF gene deleted This work .DELTA.spo0A .DELTA.pyrF Derived
from ATCC 25755, with spo0A and pyrF This work genes deleted
WT(pJZ98-Pcat1- Derived from ATCC 25755, harboring plasmid This
work adhE1) pJZ98-Pcat1-adhE1 WT(pJZ98-Pcat1- Derived from ATCC
25755, harboring plasmid This work adhE2) pJZ98-Pcat1-adhE2
.DELTA.cat1::adhE1 Derived from ATCC 25755, cat1 was replaced with
This work adhE1 .DELTA.cat1::adhE2 Derived from ATCC 25755, cat1
was replaced with This work adhE2 Plasmids pYW34-BtgZI CAK1 ori,
ColE1 ori, Amp.sup.R, Erm.sup.R, Plac-Cas9, (Wang et gRNA al.,
2016) pJZ23-Cas9 pYW34-BtgZI derivative; pBP1 ori, ColE1 ori, This
work Amp.sup.R, Cm.sup.R, TraJ, Plac-Cas9, gRNA pJZ23-Cas9-spo0A
pJZ23-Cas9 derivative; 20 nt-gRNA targeting on This work spo0A; two
homology arms (~1 kb each) pJZ58-nCas9 pJZ23-Cas9 derivative;
Plac-nCas9 This work pJZ58-nCas9-spo0A pJZ58-nCas9 derivative; 20
nt-gRNA targeting on This work spo0A; two homology arms (~1 kb
each) pMTL82151 pBP1 ori, Cm.sup.R, ColE1 ori, TraJ (Heap et al.,
2009) pWH36-AsCpf1 pMTL82151 derivative; Plac-AsCpf1 This work
pJZ60-AsCpf1- pWH36-AsCpf1 derivative; 23 nt-crRNA targeting This
work spo0A on spo0A; two homology arms (-1 kb each) pIF-1 pMTL82151
derivative; protospacer Array1-17 This work flanked by 5' PAM
sequence: 5'-CATCT-3' pIF-2 pMTL82151 derivative; protospacer
Array1-17 This work flanked by 5' PAM sequence: 5'-CATCA-3' pIF-3
pMTL82151 derivative; protospacer Array1-17 This work flanked by 3'
PAM sequence: 5'-AGGAT-3' pIF-4 pMTL82151 derivative; protospacer
Array1-17 This work flanked by 3' PAM sequence: 5'-CGGAT-3' pIF-5
pMTL82151 derivative; protospacer Array1-17 This work flanked by 5'
PAM sequence: 5'-AATTG-3' pIF-6 pMTL82151 derivative; protospacer
Array1-17 This work flanked by 5' PAM sequence: 5'-TTTCA-3' pIF-7
pMTL82151 derivative; protospacer Array1-17 This work flanked by 5'
PAM sequence: 5'-TATCT-3' pIF-8 pMTL82151 derivative; protospacer
Array2-1 This work flanked by 5' PAM sequence: 5'-CATCT-3' pIF-9
pMTL82151 derivative; protospacer Array2-1 This work flanked by 5'
PAM sequence: 5'-CATCA-3' pIF-10 pMTL82151 derivative; protospacer
Array2-1 This work flanked by 3' PAM sequence: 5'-AGGAT-3' pIF-11
pMTL82151 derivative; protospacer Array2-1 This work flanked by 3'
PAM sequence: 5'-CGGAT-3' pIF-12 pMTL82151 derivative; protospacer
Array2-1 This work flanked by 5' PAM sequence: 5'-AATTG-3' pIF-13
pMTL82151 derivative; protospacer Array2-1 This work flanked by 5'
PAM sequence: 5'-TTTCA-3' pIF-14 pMTL82151 derivative; protospacer
Array2-1 This work flanked by 5' PAM sequence: 5'-TATCT-3' pIF-15
pMTL82151 derivative; protospacer Array2-1 This work flanked by 5'
PAM sequence: 5'-GTCA-3' pIF-16 pMTL82151 derivative; protospacer
Array2-1 This work flanked by 5' PAM sequence: 5'-CTCA-3' pIF-17
pMTL82151 derivative; protospacer Array2-1 This work flanked by 5'
PAM sequence: 5'-AACA-3' pIF-18 pMTL82151 derivative; protospacer
Array2-1 This work flanked by 5' PAM sequence: 5'-AGCA-3' pIF-19
pMTL82151 derivative; protospacer Array2-1 This work flanked by 5'
PAM sequence: 5'-ACCA-3' pIF-20 pMTL82151 derivative; protospacer
Array2-1 This work flanked by 5' PAM sequence: 5'-ATGA-3' pIF-21
pMTL82151 derivative; protospacer Array2-1 This work flanked by 5'
PAM sequence: 5'-ATTA-3' pIF-22 pMTL82151 derivative; protospacer
Array2-1 This work flanked by 5' PAM sequence: 5'-ATAA-3' pIF-23
pMTL82151 derivative; protospacer Array2-1 This work flanked by 5'
PAM sequence: 5'-ATCC-3' pIF-24 pMTL82151 derivative; protospacer
Array2-1 This work flanked by 5' PAM sequence: 5'-ATCG-3'
pJZ69-leader- pMTL82151 derivative; Type I-B CRISPR genome This
work 38spo0A editing plasmid containing the native leader and
terminator sequences, the synthetic CRISPR array possessed a 38 nt
spacer1 (5'- ATACCGTTTTCTTGCTCTCACTACTATTAGCTA TATCA-3') targeting
on the spo0A gene, and two homology arms (~1 kb each) for spo0A
deletion pJZ74-Plac-38spo0A Same as pJZ69-leader-38spo0A, except
that a This work lactose inducible promoter (instead of the native
leader sequence) was used to drive the transcription of the CRISPR
array pJZ75-Plac-38spo0A Same as pJZ74-Plac-38spo0A, except that
the 38-nt This work spacer1 was replaced with the 38-nt spacer2
(5'- GCAACCATAGCTATAAATTCTGAATTTGTTGG TTTACC-3') pJZ76-Para- Same
as pJZ74-Plac-38spo0A, except that the This work 38spo0A lactose
inducible promoter was replaced with an arabinose inducible
promoter pJZ74-Plac-10spo0A Same as pJZ74-Plac-38spo0A, except that
the 38-nt This work spacer1 was replaced with the 10-nt spacer1
(5'- ATACCGTTTT-3') pJZ74-Plac-20spo0A Same as pJZ74-Plac-38spo0A,
except that the 38-nt This work spacer1 was replaced with the 20-nt
spacer1 (5'- ATACCGTTTTCTTGCTCTCA-3') pJZ74-Plac-30spo0A Same as
pJZ74-Plac-38spo0A, except that the 38-nt This work spacer1 was
replaced with the 30-nt spacer1 (5'-
ATACCGTTTTCTTGCTCTCACTACTATTAG-3') pJZ74-Plac-50spo0A Same as
pJZ74-Plac-38spo0A, except that the 38-nt This work spacer1 was
replaced with the 50-nt spacer1 (5'-
ATACCGTTTTCTTGCTCTCACTACTATTAGCTA TATCATTATTAAACATT-3')
pJZ77-Plac-30spo0A Same as pJZ74-Plac-30spo0A, except that ~300 bp
This work homology arms were used (instead of ~1 kb arms)
pJZ77-Plac-30pyrF Same as pJZ74-Plac-38spo0A, except that the 38-nt
This work spacer1 was replaced with the 30-nt spacer3 (5'-
TTGGATGTTCTTATAAGGACAAATACTCCT-3') targeting on the pyrF gene and
the homology arms for spo0A deletion were replaced with the
homology arms (~300 bp each .times.2) for pyrF deletion pJZ77-Plac-
Combined pJZ77-Plac-30spo0A and pJZ77-Plac- This work
30spo0A/30pyrF 30pyrF, including the 30-nt spacer1 targeting on the
spo0A gene, the 30-bp spacer3 targeting on the pyrF gene, the
homology arms (~300 bp each .times.2) for spo0A deletion and the
homology arms (~300 bp each .times.2) for pyrF deletion pJZ86-Plac-
Same as pJZ74-Plac-38spo0A, except that the 38-nt This work
34pta/ack spacer1 was replaced with the 34-nt spacer4 (5'-
GATTGTGCTGTAAATCCTGTACCTAATACTGA AC-3') targeting on the pta-ack
operon and the homology arms for spo0A deletion were replaced with
the homology arms (~500 bp each .times.2) for pta- ack deletion
pJZ86-Plac- pJZ86-Plac-34pta/ack derivative; adhE1 was This work
34pta/ack(adhE1) inserted between the two homology arms pJZ86-Plac-
pJZ86-Plac-34pta/ack derivative; adhE2 was This work
34pta/ack(adhE2) inserted between the two homology arms
pJZ95-Plac-34cat1 Same as pJZ74-Plac-38spo0A, except that the 38-nt
This work spacer1 was replaced with the 34-nt spacer5 (5'-
CTTGTAGAAGATGGATCAACCCTACAACTTG GTA-3'; SEQ ID NO: 4) targeting on
the cat1 gene and the homology arms for spo0A deletion were
replaced with the homology arms (~500 bp each .times.2) for cat1
deletion pJZ95-Plac- pJZ95-Plac-34cat1 derivative; adhE1 was
inserted This work 34cat1(adhE1) between the two homology arms
pJZ95-Plac- pJZ95-Plac-34cat1 derivative; adhE2 was inserted This
work 34cat1(adhE2) between the two homology arms pJZ98-Pcat1
pMTL82151 derivative; containing cat1 promoter This work
pJZ98-Pcat1-adhE1 pJZ98-Pcat1 derivative; plasmid-based adhE1 gene
This work overexpression driven by the cat1 gene promoter
pJZ98-Pcatl-adhE2 pJZ98-Pcat1 derivative; plasmid-based adhE2 gene
This work overexpression driven by the cat1 gene promoter
TABLE-US-00005 TABLE 4 Primers used in Example 1 Primers (pair)
Sequences spo0A deletion using CRISPR-Cas9 or CRISPR-nCas9 system
Cm marker 5'-ACAATTGAATTTAAAAGAAACCGATAGGCCGGCCAGTGGGCAA GTTG-3'
(SEQ ID NO: 26) 5'-CTTTAGTAACGTGTAACTTTCCAAATGGAGTTTAAACTTAGGG
TAAC-3' (SEQ ID NO: 27) in vitro Cas9
5'-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGG nuclease
ACTAGCCTTATTTTAACTT GCTATTTCTAGCTCTAAAAC-3' double (SEQ ID NO: 28)
digestion of 5'-AGAAATTAATACGACTCACTATAGGGATACTAAAACTGAATTGA CAK1
TTGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGG-3' (SEQ ID NO: 29)
5'-AGAAATTAATACGACTCACTATAGGGAGTGCAAAAAAAGATATA ATGTTTTAGAGCTAGAAAT
AGCAAGTTAAAATAAGG-3' (SEQ ID NO: 30) pBP1 replicon
5'-CGAACACGAACCGTCTTATCTCCCATTGTTCTGAATCCTTAGCT AATGG-3' (SEQ ID
NO: 31) 5'-TAATGACCCCGAAGCAGGGGGCCCAATGAATTTGTAAATAAACC ACAAAC-3'
(SEQ ID NO: 32) TraJ 5'-GTAATACTAAAACTGAATTGATTCCTGCTTCGGGGTCATTATA
G-3' (SEQ ID NO: 33)
5'-ATCAAGTAAATAAACCAAGTATATAAGGGCCCGATCGGTCTTGC CTTGCTCGTCG-3' (SEQ
ID NO: 34) PsRNA + 20 nt
5'-AAAGTTAAAAGAAGAAAATAGAAATATAATCTTTAATTTGAAAA protospacer
GATTTAAG-3' (SEQ ID NO: 35) sequence
5'-TTGCTATTTCTAGCTCTAAAACCGCTACTTCAATAGCATGTCAT Homology
GGTGGAATGATAAGGG-3' (SEQ ID NO: 36) arms (~1 kb
5'-CTTTGTGATATGACTAATAATTAGCGGCCGCCTCAGGGTGTATT each) AGTTGTAG-3'
(SEQ ID NO: 37) 5'-GTTAACCATTGATATCACTTTAATATTTTACTCCCCTTTTAT T-3'
(SEQ ID NO: 38) 5'-AATAAAAGGGGAGTAAAATATTAAAGTGATATCAATGGTTAA C-3'
(SEQ ID NO: 39) 5'-ATCCACTAGTAACCATCACACTGGCGGCCGCGACCAATACTGAA
CTATGACC-3' (SEQ ID NO: 40) Plac-nCas9
5'-CACCGACGAGCAAGGCAAGACCGATCGGGCCCTTATATACTTGG TTTATTTACTTG-3'
(SEQ ID NO: 41) 5'-CCTATTGAGTATTTCTTATCCATTTCAGCCCTCCTGTGAAATT G-3'
(SEQ ID NO: 42) 5'-CAATTTCACAGGAGGGCTGAAATGGATAAGAAATACTCAATAG G-3'
(SEQ ID NO: 43) 5'-GATAAATTTATAAAATTCTTCTTGGC-3' (SEQ ID NO: 44)
spo0A deletion using CRISPR-AsCpf1 system Plac-AsCpf1
5'-GGAAACAGCTATGACCGCGGCCGCTGTATCTTATATACTTGGTT TATTTACTTGATTATT-3'
(SEQ ID NO: 45) 5'-TGGTAGAGATTGGTGAAGCCTTCAAACTGTGTCATTTCAGCCCT
CCTGTGAAATTGTTATCCG CTCACAA-3' (SEQ ID NO: 46)
5'-TTGTGAGCGGATAACAATTTCACAGGAGGGCTGAAATGACACAG TTTGAAGGCTTCACCAAT
CTCTACCA-3' (SEQ ID NO: 47)
5'-GGGTACCGAGCTCGAATTCGTAATCATGGTTTAGTTTCTCAGTT CTTGAATGTAGGCCAG-3'
(SEQ ID NO: 48) PsRNA-
5'-GATTACGAATTCGAGCTCGGTACCCGGGATAATCTTTAATTTGA crRNA
AAAGATTTAAG-3' (SEQ ID NO: 49)
5'-TTAGCTGAAAGCACGATTACTCTCGGATCTACAAGAGTAGAAAT TAATGGTGG-3' (SEQ
ID NO: 50) Homology 5'-GATCCGAGAGTAATCGTGCTTTCAGCTAATTTCTACTCTTGTAG
arms (~1 kb ATCTCAGGGTGTATTAGTTG TAG-3' (SEQ ID NO: 51) each)
5'-CCATGGACGCGTGACGTCGACTCTAGAGGACCAATACTGAACTA TGACC-3' (SEQ ID
NO: 52) spo0A deletion using endogenous Type I-B CRISPR-Cas system
Leader + 38-nt 5'-CTGTATCCATATGACCATGATTACGTAAGATCGTAGCAGATAAG
spacer1 + GAT-3' (SEQ ID NO: 53) terminator
5'-GCTAATAGTAGTGAGAGCAAGAAAACGGTATATTTAAATACATC
TCATGTTAAGGTTCAACCTGTGTAAAATAGCCATTC-3' (SEQ ID NO: 54)
5'-TTTCTTGCTCTCACTACTATTAGCTATATCAGTTGAACCTTAAC
ATGAGATGTATTTAAATCCCATAGAAGCTCTATACT-3' (SEQ ID NO: 55)
5'-CTACAACTAATACACCCTGAGGGTACCTGGAGATATAATAAGCT ATGCC-3' (SEQ ID
NO: 56) Homology 5'-CATGATTACGAATTCGAGCTCGGTACCCTCAGGGTGTATTAGTT
arms (~1 kb GTAG-3' (SEQ ID NO: 57) each)
5'-GTTAACCATTGATATCACTTTAATATTTTACTCCCCTTTTAT T-3' (SEQ ID NO: 58)
5'-AATAAAAGGGGAGTAAAATATTAAAGTGATATCAATGGTTAA C-3' (SEQ ID NO: 59)
5'-TGGACGCGTGACGTCGACTCTAGAGGACCAATACTGAACTATGA CC-3' (SEQ ID NO:
60) Plac + 38-nt 5'-CTGTATCCATATGACCATGATTACGGATTGGGCCCTTATATACT
spacer1 + TGG-3' (SEQ ID NO: 61) terminator
5'-GCAAGAAAACGGTATATTTAAATACATCTCATGTTAAGGTTCAA CTTCAGCCCTCCTGTGAAA
TTG-3' (SEQ ID NO: 62)
5'-CATGAGATGTATTTAAATATACCGTTTTCTTGCTCTCAC-3' (SEQ ID NO: 63)
5'-CTACAACTAATACACCCTGAGGGTACCTGGAGATATAATAAGCT ATGCC-3' (SEQ ID
NO: 64) Plac + 38-nt
5'-CCAACAAATTCAGAATTTATAGCTATGGTTGCATTTAAATACAT spacer2 +
CTCATGTTAAGGTTCAACTTCAGCCCTCCTGTGAAATTG-3' (SEQ terminator ID NO:
65) 5'-ATAGCTATAAATTCTGAATTTGTTGGTTTACCGTTGAACCTTAA
CATGAGATGTATTTAAATCCCATAGAAGCTCTATACT-3' (SEQ ID NO: 66) Para +
38-nt 5'-CTGTATCCATATGACCATGATTACGTTATGAAAGCGATTACCTA spacer1 +
TAT-3' (SEQ ID NO: 67) terminator
5'-GCAAGAAAACGGTATATTTAAATACATCTCATGTTAAGGTTCAA
CAATATTCCTCCTAAATTTATAATC-3' (SEQ ID NO: 68) Plac + 10-nt
5'-GGTTCAACAAAACGGTATATTTAAATACATCTCATGTTAAGGTT spacer1 +
CAACTTCAGCCCTCCTGTG AAATTG-3' (SEQ ID NO: 69) terminator
5'-ATTTAAATATACCGTTTTGTTGAACCTTAACATGAGATGTATTT
AAATCCCATAGAAGCTCTATACT-3' (SEQ ID NO: 70) Plac + 20-nt
5'-CAACTGAGAGCAAGAAAACGGTATATTTAAATACATCTCATGTT spacer1 +
AAGGTTCAACTTCAGCCCT CCTGTGAAATTG-3' (SEQ ID terminator NO: 71)
5'-AAATATACCGTTTTCTTGCTCTCAGTTGAACCTTAACATGAGAT
GTATTTAAATCCCATAGAAG CTCTATACT-3' (SEQ ID NO: 72) Plac + 30-nt
5'-CTAATAGTAGTGAGAGCAAGAAAACGGTATATTTAAATACATCT spacer1 +
CATGTTAAGGTTCAACTTC AGCCCTCCTGTGAAATTG-3' (SEQ terminator ID NO:
73) 5'-ATACCGTTTTCTTGCTCTCACTACTATTAGGTTGAACCTTAACA
TGAGATGTATTTAAATCCCA TAGAAGCTCTATACT-3' (SEQ ID NO: 74) Plac +
50-nt 5'-GATATAGCTAATAGTAGTGAGAGCAAGAAAACGGTATATTTAAA spacer1 +
TACATCTCATGTTAAGGTTCAACTTCAGCCCTCCTGTGAAATTG-3' terminator (SEQ ID
NO: 75) 5'-TTGCTCTCACTACTATTAGCTATATCATTATTAAACATTGTTGA
ACCTTAACATGAGATGTATTTAAATCCCATAGAAGCTCTATACT-3' (SEQ ID NO: 76)
spo0A and pyrF double deletion using endogenous Type I-B CRISPR-Cas
system spo0A deletion
5'-CATGATTACGAATTCGAGCTCGGTACCGTTCAAGGTATGAGTGG (arms, ~300
AAGTCC-3' (SEQ ID NO: 77) bp each)
5'-TGGACGCGTGACGTCGACTCTAGAGACATCTTCTATATATCTGC pyrF deletion
AAAATAGCTTC-3' (SEQ ID NO: 78) (30-nt spacer)
5'-CCTGACTCTAGAGTCGACGTCACGCGTCGATTGGGCCCTTATAT ACTTGG-3' (SEQ ID
NO: 79) 5'-AGGAGTATTTGTCCTTATAAGAACATCCAAATTTAAATACATCT
CATGTTAAGGTTCAACTTCAGCCCTCCTGTGAAATTG-3' (SEQ ID NO: 80)
5'-TTGGATGTTCTTATAAGGACAAATACTCCTGTTGAACCTTAACA
TGAGATGTATTTAAATCCCATAGAAGCTCTATACT-3' (SEQ ID NO: 81)
5'-CGACGTTGTAAAACGACGGCCAGTGCCATGGAGATATAATAAGC TATGCC-3' (SEQ ID
NO: 82) pyrF deletion
5'-CTGTATCCATATGACCATGATTACGGCTATATTGGGTTTCATAG (arms, ~300 ATCC-3'
(SEQ ID NO: 83) bp each)
5'-GCACACTCTGCATAGTCTGTGTAAGTATCCAGGCCTACACATA C-3' (SEQ ID NO: 84)
5'-GTATGTGTAGGCCTGGATACTTACACAGACTATGCAGAGTGTG C-3' (SEQ ID NO: 85)
5'-TGGACGCGTGACGTCGACTCTAGAGTAGTTCCATTTCCAACTAC CTG-3' (SEQ ID NO:
86) spo0A + pyrF 5'-CTGTATCCATATGACCATGATTACGCCCGGGGATTGGGCCCTTA
deletion TATACTTGG-3' (SEQ ID NO: 87) ((30 + 30) nt
5'-GGAGTATTTGTCCTTATAAGAACATCCAAATTTAAATACATCTC spacer)
ATGTTAAGGTTCAACTTCAG CCCTCCTGTGAAATTG-3' (SEQ ID NO: 88)
5'-GGATGTTCTTATAAGGACAAATACTCCTGTTGAACCTTAACATG
AGATGTATTTAAATATACCG TTTTCTTGCTCTCAC-3' (SEQ ID NO: 89)
5'-CTACAACTAATACACCCTGAGGGTACCTGGAGATATAATAAGCT ATGCC-3' (SEQ ID
NO: 90) spo0A + pyrF
5'-CATGATTACGAATTCGAGCTCGGTACCGCTATATTGGGTTTCAT deletion AGATCC-3'
(SEQ ID NO: 91) ((~300 + ~300)
5'-GGACTTCCACTCATACCTTGAACTAGTTCCATTTCCAACTACCT bp arms) G-3' (SEQ
ID NO: 92) 5'-CAGGTAGTTGGAAATGGAACTAGTTCAAGGTATGAGTGGAAGTC C-3'
(SEQ ID NO: 93) 5'-TGGACGCGTGACGTCGACTCTAGAGACATCTTCTATATATCTGC
AAAATAGCTTC-3' (SEQ ID NO: 94) pta/ack deletion (or replaced by
adhE1/adhE2) using endogenous Type I-B CRISPR-Cas system Plac +
34-nt 5'-CTGTATCCATATGACCATGATTACGGATTGGGCCCTTATATACT spacer4 +
TGG-3' (SEQ ID NO: 95) terminator
5'-AGTATTAGGTACAGGATTTACAGCACAATCATTTAAATACATCT CATGTTAAGGTTCAACTTC
AGCCCTCCTGTGAAATTG-3' (SEQ ID NO: 96)
5'-GTGCTGTAAATCCTGTACCTAATACTGAACGTTGAACCTTAACA
TGAGATGTATTTAAATCCCATAGAAGCTCTATACT-3' (SEQ ID NO: 97)
5'-GTCGACTCTAGAGGATCCCCGGGTACCTGGAGATATAATAAGCT ATGCC-3' (SEQ ID
NO: 98) Homology 5'-GGCATAGCTTATTATATCTCCAGGTACGTATCAACTACGCCTAA
arms (~500 bp ATTCTCC-3' (SEQ ID NO: 99) each)
5'-TAGGCTGTTCAGGGATCCCCGGGTACCTTTCGTTTCTCCCTTCA AGAT-3' (SEQ ID NO:
100) 5'-GGAGAAACGAAAGGTACCCGGGGATCCCTGAACAGCCTATGGAA GACC-3' (SEQ
ID NO: 101) 5'-TGGACGCGTGACGTCGACTCTAGAGCACCGTCAATTGCACATAC AC-3'
(SEQ ID NO: 102) adhE1
5'-TATCTTGAAGGGAGAAACGAAAGGTACATGAAAGTCACAACAGT AAAGG-3' (SEQ ID
NO: 103) 5'-TTATGGTCTTCCATAGGCTGTTCAGGGTTGAAATATGAAGGTTT AAGGTTG-3'
(SEQ ID NO: 104) adhE2
5'-TATCTTGAAGGGAGAAACGAAAGGTACATGAAAGTTACAAATCA AAAAG-3' (SEQ ID
NO: 105) 5'-TTATGGTCTTCCATAGGCTGTTCAGGTTAAAATGATTTTATATA GATATCC-3'
(SEQ ID NO: 106) cat1 deletion (or replaced by adhE1/adhE2) using
endogenous Type I-B CRISPR-Cas system Plac + 34-nt
5'-CTGTATCCATATGACCATGATTACGGATTGGGCCCTTATATACT spacer5 + TGG-3'
(SEQ ID NO: 107) terminator
5'-AGTTGTAGGGTTGATCCATCTTCTACAAGATTTAAATACATCTC
ATGTTAAGGTTCAACTTCAGCCCTCCTGTGAAATTG-3' (SEQ ID NO: 108)
5'-GTAGAAGATGGATCAACCCTACAACTTGGTAGTTGAACCTTAAC ATGAGATGTATTTAAATCC
CATAGAAGCTCTATACT-3' (SEQ ID NO: 109)
5'-GTCGACTCTAGAGGATCCCCGGGTACCTGGAGATATAATAAGCT ATGCC-3' (SEQ ID
NO: 110) Homology 5'-GGCATAGCTTATTATATCTCCAGGTACACCCATGCTGCAAAGCA
arms (~500 bp AGTT-3' (SEQ ID NO: 111) each)
5'-TGAGAAAGCTAAGGATCCCCGGGTACCAAAAACCACCCTTTCAT AAATT-3' (SEQ ID
NO: 112) 5'-GGGTGGTTTTTGGTACCCGGGGATCCTTAGCTTTCTCAAAAGAT ATTTT-3'
(SEQ ID NO: 113) 5'-TGGACGCGTGACGTCGACTCTAGAGCCATATGCGGTGGTTATC
AAC-3' (SEQ ID NO: 114) adhE1
5'-AATTTATGAAAGGGTGGTTTTTGGTACATGAAAGTCACAACAGT AAAGG-3' (SEQ ID
NO: 115) 5'-TTAAAAATATCTTTTGAGAAAGCTAAGGTTGAAATATGAAGGTT
TAAGGTTG-3' (SEQ ID NO: 116) adhE2
5'-AATTTATGAAAGGGTGGTTTTTGGTACATGAAAGTTACAAATCA AAAAG-3' (SEQ ID
NO: 117) 5'-TTAAAAATATCTTTTGAGAAAGCTAAGTTAAAATGATTTTATAT
AGATATCC-3' (SEQ ID NO: 118)
Plasmid based adhE1/adhE2 overexpression cat1 promoter
5'-CTGTATCCATATGACCATGATTACGGTAGACTTTAAGGATGGAA CC-3' (SEQ ID NO:
119) 5'-TCGACTCTAGAGGATCCCCGGGTACCGAATTCTGTCGACTGCGA
TGAGCTAGGTCAGTAAAA ACCACCCTTTCATAAATT-3' (SEQ ID NO: 120) adhE1
5'-ATATAATTTATGAAAGGGTGGTTTTTATGAAAGTCACAACAGTA AAGG-3' (SEQ ID NO:
121) 5'-CGACTCTAGAGGATCCCCGGGTACCGAATTCGTTGAAATATGAA
GGTTTAAGGTTG-3' (SEQ ID NO: 122) adhE2
5'-ATATAATTTATGAAAGGGTGGTTTTTATGAAAGTTACAAATCAA AAAG-3' (SEQ ID NO:
123) 5'-CGACTCTAGAGGATCCCCGGGTACCGGTAACCTTAAAATGATTT
TATATAGATATCC-3' (SEQ ID NO: 124) Mutant detection spo0A deletion
5'-TGTTCCTGTAGGATCAGTATC-3' (SEQ ID NO: 125)
5'-GGACTGTACCTCTGGTAGTTC-3' (SEQ ID NO: 126) pyrF deletion
5'-GTTGAAAGACAGCTATATCTTGG-3' (SEQ ID NO: 127)
5'-ATGCCATGTGATTCTCCATAG-3' (SEQ ID NO: 128) Pta-ack
5'-TCTATACCTTCAGATACTTCTGG-3' (SEQ ID NO: 129) deletion
5'-CTCACCTCTATACATTAGCCAC-3' (SEQ ID NO: 130) cat1 deletion
5'-GCCATTAAGTACAAATGAGATAG-3' (SEQ ID NO: 131)
5'-GCCATTAAGTACAAATGAGATAG-3' (SEQ ID NO: 132)
Discussion
Within the past few years, CRISPR-Cas, the adaptive immune system
from bacteria and archaea, has been repurposed for versatile genome
editing and transcriptional regulation in various strain. However,
so far, the majority of such applications are based on the Type II
CRISPR-Cas9 system derived from S. pyogenes.
Due to the unique feature of the chromosome of prokaryotic cells,
the expression of the heterologous Cas9 is highly toxic, thus
leading to poor transformation efficiency and failure of genome
editing. Recently, the type V CRISPR-Cpf1 system has also been
exploited for genome editing purposes. It has advantages over the
CRISPR-Cas9 system due to its smaller size of the effector protein
(Cpf1) and the more compact RNA guide (crRNA). Although the
toxicity of Cpf1 is much lower than that of Cas9 as demonstrated in
specific strains, remarkable decrease in transformation efficiency
is still observed with the expression of Cpf1 in the host.
Therefore, it is challenging to carry out genome editing with
CRISPR-Cas9/Cpf1 systems in microorganisms with low DNA
transformation efficiencies.
In this work, after many unsuccessful attempts for genome editing
with the CRISPR-Cas9 or CRISPR-AsCpf1 systems, we successfully
repurposed the Type I-B CRISPR-Cas system of C. tyrobutyricum as an
efficient genome editing tool for this microorganism.
In silico analysis of the CRISPR array in C. tyrobutyricum
identified only one spacer sequence that can match protospacers
from phage (prophage) of Clostridium and Geobacillus (FIG. 1B).
However, we hypothesized that, due to the possible horizontal
transferring property of CRISPR-Cas loci between closely-related
strain, the Type I-B CRISPR-Cas systems from different Clostridium
strain could be very similar and share similar/same PAMs and direct
repeat sequences. Indeed, our subsequent in silico analysis
demonstrated high homology between the CRISPR array in C.
tyrobutyricum and that in C. pasteurianum. Therefore, the three PAM
sequences from C. pasteurianum along with the putative PAMs
identified in C. tyrobutyricum were employed to assess the activity
of the endogenous CRISPR-Cas system of C. tyrobutyricum. The in
vivo plasmid interference assay revealed that the Cas protein in C.
tyrobutyricum had high affinity to the 5' adjacent PAM sequences
TCA and TCG (FIG. 2B). These results verified our hypothesis that
the Type I-B CRISPR-Cas system from C. tyrobutyricum shares the
same PAM sequence (TCA) as that in C. pasteurianum, as well as
those in Clostridium tetani and C. thermocellum.
In attempt for the genome editing with the endogenous CRISPR-Cas
system, initially, the native leader sequence was used as the
promoter to drive the transcription of the synthetic CRISPR array.
However, no transformants were obtained, likely due to the toxicity
of the endogenous CRISPR-Cas system when it was instantly
expressed. A lactose inducible promoter was employed to replace the
leader sequence to drive the expression of the CRISPR-Cas system,
resulting in an overall transformation efficiency of 1.7 CFU/mL
donor (FIG. 3C). This transformation efficiency is still low, but
is enough to enable us to obtain desirable mutants with a high
editing efficiency. With this, we demonstrated that the inducible
expression of the endogenous CRISPR-Cas array is achievable
(although the configuration of the original native leader sequence
for the CRISPR array regulation was complex) and effective to
realize efficient genome editing in the host microorganism. It is
also worthwhile to point out that, the same inducible promoter was
also used to drive the expression of Cas9, nCas9 or AsCpf1 proteins
to achieve genome editing for the same microorganism, however no
successful transformation was achieved with any of the plasmids
containing these heterologous nuclease (or nickase) proteins. This
confirmed that the toxicity of the endogenous CRISPR-Cas system is
much lower than that of heterologous CRISPR-Cas9/nCas9/AsCpf1
systems and thus more implementable for genome editing purposes
(FIGS. 3B & 3C).
Although the markerless genome engineering platform was developed,
and high editing efficiency could be obtained, the transformation
efficiency was still low which would restrict the application of
the genome editing platform in C. tyrobutyricum. The length of
spacers identified from the CRISPR Array1 and Array2 are not all
the same (ranging from 34-38 nt). We reasoned that the length of
the spacer might have an impact on the transformation efficiency
and/or genome editing efficiency. Therefore, various lengths of
spacers were systematically evaluated in the developed CRISPR-Cas
system in the context for spo0A deletion. Results indicated that,
the transformation was not successful when the spacer .ltoreq.20 nt
was used, suggesting possible severe off-target effects (FIG. 3C).
Spacers ranging from 30 to 50 nt can be used for targeting purposes
for the successful genome editing. Comparatively, when shorter
spacers were used, the genome editing efficiency was slightly
decreased (for 30-nt spacer, an editing efficiency of 93.3% was
obtained based on the large colonies), but meanwhile the
transformation efficiency was dramatically enhanced (by
approximately 500-fold for the 30-nt spacer). Therefore, depending
on the different genome editing purposes, one can make a tradeoff
between the transformation efficiency and genome editing efficiency
by using a spacer of an appropriate length. Briefly, based on the
above results for the deletion of spo0A particularly, spacers of
30-38 nt seems good options. It should be pointed out that, one of
the advantages with such an endogenous CRISPR-Cas system comparing
to the type II CRISPR-Cas9 system for genome editing is that, the
employment of the longer spacer sequence (30-38 nt vs. 20 nt for
the spCRISPR-Cas9) can abate the potential off-target effect.
Apparently, the longer the spacer sequence is, the more specific
the targeting of the crRNA. For eukaryotic cells, the off-target
effect can lead to unspecific mutations on the chromosome, which is
highly problematic for various applications. This won't occur for
the prokaryotic cells due to their inefficient endogenous
nonhomologous end-joining (NHEJ) capability for the automatic DNA
repairing. However, the off-target effect in prokaryotic cells can
lead to cell death and thus failure of genome editing.
In this study, multiplex genome editing was achieved by using the
endogenous CRISPR-Cas system of C. tyrobutyricum (FIG. 4A). A
synthetic CRISPR array carrying two spacers was used for the
chromosome targeting to delete spo0A and pyrF simultaneously,
yielding an editing efficiency of up to 100% (FIG. 4C). To date,
this is the first success for multiplex genome editing in
microorganisms with underdeveloped genome engineering tools such as
Clostridium.
C. tyrobutyricum is a natural hyper-butyrate producer, which has
been engineered for butanol production previously. The cat1 gene is
believed to be the essential gene for butyrate production in C.
tyrobutyricum, and the deletion of cat1 was not previously
achievable. In this study, based on the developed CRISPR-Cas genome
engineering system, we successfully replaced the cat1 gene with
adhE1/adhE2. In this way, the butyrate production in C.
tyrobutyricum was almost eliminated and the microorganism was
converted into a hyper-butanol producer (FIG. 5). Previous studies
have demonstrated that the lower temperature is beneficial to
enhance the butanol tolerance of host strains, which may be because
of the change of cell membrane composition and fluidity under lower
temperatures. Therefore, fermentations for butanol production with
the C. tyrobutyricum mutant were further carried out at lower
temperatures. At 20.degree. C., the butanol production in the
mutant .DELTA.cat1::adhE2 reached 26.2 g/L in a regular batch
fermentation. To the best of our knowledge, this is the highest
butanol production that has ever been reported in a batch
fermentation. We also investigated the butanol production of C.
beijerinckii NCIMB 8052 and C. saccharoperbutylacetonicum N1-4 at
lower temperatures (Table 5), to confirm whether carrying out
fermentations at low temperatures is a broadly applicable mechanism
to achieve high butanol production with other strains as well.
Although the butanol production of these two solventogenic
clostridia was increased at lower temperatures, the increment was
far lower than that obtained with mutant .DELTA.cat1::adhE2,
indicating that C. tyrobutyricum has much greater potential and
thus is a more favorable host for butanol production. Furthermore,
there is no acetone production in the fermentation of
.DELTA.cat1::adhE2 as seen in the ABE fermentation; butanol,
ethanol and acetate are the only primary end products. This on one
hand simplifies the downstream recovery process; on the other,
these end products could be further upgraded to high-value
biochemicals (such as diesel, esters, etc.) through chemical or
biochemical processes.
TABLE-US-00006 TABLE 5 Summary of fermentation results for C.
beijerinckii NCIMB 8052 and C. saccharoperbutylacetonicum N1-4 at
various temperatures.sup.a Temperature Acetate Butyrate Acetone
Ethanol Butanol Total ABE ABE yield Strain (.degree. C.) (g/L)
(g/L) (g/L) (g/L) (g/L) (g/L) (g/g of glucose) 8052 35 0.10 .+-.
0.01 0.59 .+-. 0.04 5.70 .+-. 0.14 0.26 .+-. 0.02 9.68 .+-. 0.33
15.64 0.38 8052 30 0.13 .+-. 0.01 0.16 .+-. 0.05 6.31 .+-. 0.10
0.32 .+-. 0.03 9.96 .+-. 0.18 16.59 0.36 8052 25 0.39 .+-. 0.02
1.28 .+-. 0.13 3.33 .+-. 0.50 0.40 .+-. 0.03 9.71 .+-. 0.12 13.44
0.35 8052 20 0.22 .+-. 0.02 2.13 .+-. 0.13 3.75 .+-. 0.06 0.24 .+-.
0.01 11.12 .+-. 0.29 15.11 0.31 N1-4 30 0.94 .+-. 0.14 1.88 .+-.
0.28 5.89 .+-. 0.12 1.02 .+-. 0.15 17.10 .+-. 0.25 24.01 0.35 N1-4
25 1.23 .+-. 0.12 0.53 .+-. 0.05 4.21 .+-. 0.21 2.08 .+-. 0.04
18.07 .+-. 0.97 24.36 0.42 N1-4 20 0.41 .+-. 0.04 0.85 .+-. 0.08
4.10 .+-. 0.60 0.71 .+-. 0.01 18.09 .+-. 0.78 22.58 0.41
.sup.aValues are based on at least two independent replicates.
SEQUENCE LISTINGS
1
1341291DNAClostridium tyrobutyricum 1taagatcgta gcagataagg
attttgtcac aatcataaaa cttataaatg atagttgctt 60tgaggaggaa actttaggta
taaatgataa aaatactgaa aacttgatac tttgaatttt 120ccaacctgtt
tgctatttag aatcacttca atttatttag aatcaatggg ttacgttatt
180tcttataaaa tatatgcata ataaaaattg gttggaaaaa attcagcgaa
aacctttatt 240tatatgcttt caaagcttat aatgaaatta aagaatggct
attttacaca g 291230DNAClostridium tyrobutyricum 2gttgaacctt
aacatgagat gtatttaaat 303277DNAClostridium tyrobutyricum
3aatcaaacat tttaattaaa gagacaatta ttataaataa attggtatag aattatattg
60aataaatcta taccaatttt taatttgata tcaagccttt gttaaaatat ctgttaaacc
120caatttttct attctctttt ttatctcatt tttatctgta gtatatagtc
ccttttcttt 180cattctgtta aaatactctg cacctgcaaa agtgtatctg
ttttttctac cctctgcagt 240tttaattttg taattggcat agcttattat atctcca
2774666DNAClostridium tyrobutyricum 4taagatcgta gcagataagg
attttgtcac aatcataaaa cttataaatg atagttgctt 60tgaggaggaa actttaggta
taaatgataa aaatactgaa aacttgatac tttgaatttt 120ccaacctgtt
tgctatttag aatcacttca atttatttag aatcaatggg ttacgttatt
180tcttataaaa tatatgcata ataaaaattg gttggaaaaa attcagcgaa
aacctttatt 240tatatgcttt caaagcttat aatgaaatta aagaatggct
attttacaca ggttgaacct 300taacatgaga tgtatttaaa tataccgttt
tcttgctctc actactatta gctatatcag 360ttgaacctta acatgagatg
tatttaaata atcaaacatt ttaattaaag agacaattat 420tataaataaa
ttggtataga attatattga ataaatctat accaattttt aatttgatat
480caagcctttg ttaaaatatc tgttaaaccc aatttttcta ttctcttttt
tatctcattt 540ttatctgtag tatatagtcc cttttctttc attctgttaa
aatactctgc acctgcaaaa 600gtgtatctgt tttttctacc ctctgcagtt
ttaattttgt aattggcata gcttattata 660tctcca 666534DNAClostridium
tyrobutyricum 5cttgtagaag atggatcaac cctacaactt ggta
34620DNAClostridium tyrobutyricum 6gacatgctat tgaagtagcg
20720DNAClostridium tyrobutyricum 7taatttctac tcttgtagat
20823DNAClostridium tyrobutyricum 8ccgagagtaa tcgtgctttc agc
23938DNAClostridium tyrobutyricum 9ataccgtttt cttgctctca ctactattag
ctatatca 381038DNAClostridium tyrobutyricum 10gcaaccatag ctataaattc
tgaatttgtt ggtttacc 381110DNAClostridium tyrobutyricum 11ataccgtttt
101220DNAClostridium tyrobutyricum 12ataccgtttt cttgctctca
201330DNAClostridium tyrobutyricum 13ataccgtttt cttgctctca
ctactattag 301450DNAClostridium tyrobutyricum 14ataccgtttt
cttgctctca ctactattag ctatatcatt attaaacatt 501530DNAClostridium
tyrobutyricum 15ttggatgttc ttataaggac aaatactcct
301634DNAClostridium tyrobutyricum 16gattgtgctg taaatcctgt
acctaatact gaac 341734DNAClostridium tyrobutyricum 17cttgtagaag
atggatcaac cctacaactt ggta 341830DNAClostridium tyrobutyricum
18attgaacctt aacatgagat gtatttaaat 301936DNAClostridium
tyrobutyricum 19tggtatcacc aacttttgtc caggatatat gaggtt
362046DNAClostridium tyrobutyricum 20catctcggta tcaccaactt
ctgcccggga tatatgagat taggat 462151DNAClostridium tyrobutyricum
21catcatggta tcaccagctt ttggccggga taaatgagat tcggatcgga t
512238DNAClostridium tyrobutyricum 22gcattcagac ttgcaactgt
aactccctag tactcccc 3823124DNAClostridium tyrobutyricum
23gggttacgtt atttcttata aaatatatgc ataataaaaa ttggttggaa aaaattcagc
60gaaaaccttt atttatatgc tttcaaagct tataatgaaa ttaaagaatg gctattttac
120acag 12424127DNAClostridium tyrobutyricum 24ggcttatagg
tgtttttcta ttaaaattta cgtaagacta aaaatagctg gtaaaatttt 60tgctaaatcc
tttattttta atgaatagag cattataatt atagtaaaga atggctagtt 120ttaagta
1272530DNAClostridium tyrobutyricum 25gttgaacctt aacataggat
gtatttaaat 302647DNAClostridium tyrobutyricum 26acaattgaat
ttaaaagaaa ccgataggcc ggccagtggg caagttg 472747DNAClostridium
tyrobutyricum 27ctttagtaac gtgtaacttt ccaaatggag tttaaactta gggtaac
472883DNAClostridium tyrobutyricum 28aaaaaaagca ccgactcggt
gccacttttt caagttgata acggactagc cttattttaa 60cttgctattt ctagctctaa
aac 832980DNAClostridium tyrobutyricum 29agaaattaat acgactcact
atagggatac taaaactgaa ttgattgttt tagagctaga 60aatagcaagt taaaataagg
803080DNAClostridium tyrobutyricum 30agaaattaat acgactcact
atagggagtg caaaaaaaga tataatgttt tagagctaga 60aatagcaagt taaaataagg
803149DNAClostridium tyrobutyricum 31cgaacacgaa ccgtcttatc
tcccattgtt ctgaatcctt agctaatgg 493250DNAClostridium tyrobutyricum
32taatgacccc gaagcagggg gcccaatgaa tttgtaaata aaccacaaac
503344DNAClostridium tyrobutyricum 33gtaatactaa aactgaattg
attcctgctt cggggtcatt atag 443455DNAClostridium tyrobutyricum
34atcaagtaaa taaaccaagt atataagggc ccgatcggtc ttgccttgct cgtcg
553552DNAClostridium tyrobutyricum 35aaagttaaaa gaagaaaata
gaaatataat ctttaatttg aaaagattta ag 523660DNAClostridium
tyrobutyricum 36ttgctatttc tagctctaaa accgctactt caatagcatg
tcatggtgga atgataaggg 603752DNAClostridium tyrobutyricum
37ctttgtgata tgactaataa ttagcggccg cctcagggtg tattagttgt ag
523843DNAClostridium tyrobutyricum 38gttaaccatt gatatcactt
taatatttta ctcccctttt att 433943DNAClostridium tyrobutyricum
39aataaaaggg gagtaaaata ttaaagtgat atcaatggtt aac
434052DNAClostridium tyrobutyricum 40atccactagt aaccatcaca
ctggcggccg cgaccaatac tgaactatga cc 524156DNAClostridium
tyrobutyricum 41caccgacgag caaggcaaga ccgatcgggc ccttatatac
ttggtttatt tacttg 564244DNAClostridium tyrobutyricum 42cctattgagt
atttcttatc catttcagcc ctcctgtgaa attg 444344DNAClostridium
tyrobutyricum 43caatttcaca ggagggctga aatggataag aaatactcaa tagg
444426DNAClostridium tyrobutyricum 44gataaattta taaaattctt cttggc
264560DNAClostridium tyrobutyricum 45ggaaacagct atgaccgcgg
ccgctgtatc ttatatactt ggtttattta cttgattatt 604670DNAClostridium
tyrobutyricum 46tggtagagat tggtgaagcc ttcaaactgt gtcatttcag
ccctcctgtg aaattgttat 60ccgctcacaa 704770DNAClostridium
tyrobutyricum 47ttgtgagcgg ataacaattt cacaggaggg ctgaaatgac
acagtttgaa ggcttcacca 60atctctacca 704860DNAClostridium
tyrobutyricum 48gggtaccgag ctcgaattcg taatcatggt ttagtttctc
agttcttgaa tgtaggccag 604955DNAClostridium tyrobutyricum
49gattacgaat tcgagctcgg tacccgggat aatctttaat ttgaaaagat ttaag
555053DNAClostridium tyrobutyricum 50ttagctgaaa gcacgattac
tctcggatct acaagagtag aaattaatgg tgg 535167DNAClostridium
tyrobutyricum 51gatccgagag taatcgtgct ttcagctaat ttctactctt
gtagatctca gggtgtatta 60gttgtag 675249DNAClostridium tyrobutyricum
52ccatggacgc gtgacgtcga ctctagagga ccaatactga actatgacc
495347DNAClostridium tyrobutyricum 53ctgtatccat atgaccatga
ttacgtaaga tcgtagcaga taaggat 475480DNAClostridium tyrobutyricum
54gctaatagta gtgagagcaa gaaaacggta tatttaaata catctcatgt taaggttcaa
60cctgtgtaaa atagccattc 805580DNAClostridium tyrobutyricum
55tttcttgctc tcactactat tagctatatc agttgaacct taacatgaga tgtatttaaa
60tcccatagaa gctctatact 805649DNAClostridium tyrobutyricum
56ctacaactaa tacaccctga gggtacctgg agatataata agctatgcc
495748DNAClostridium tyrobutyricum 57catgattacg aattcgagct
cggtaccctc agggtgtatt agttgtag 485843DNAClostridium tyrobutyricum
58gttaaccatt gatatcactt taatatttta ctcccctttt att
435943DNAClostridium tyrobutyricum 59aataaaaggg gagtaaaata
ttaaagtgat atcaatggtt aac 436046DNAClostridium tyrobutyricum
60tggacgcgtg acgtcgactc tagaggacca atactgaact atgacc
466147DNAClostridium tyrobutyricum 61ctgtatccat atgaccatga
ttacggattg ggcccttata tacttgg 476266DNAClostridium tyrobutyricum
62gcaagaaaac ggtatattta aatacatctc atgttaaggt tcaacttcag ccctcctgtg
60aaattg 666339DNAClostridium tyrobutyricum 63catgagatgt atttaaatat
accgttttct tgctctcac 396449DNAClostridium tyrobutyricum
64ctacaactaa tacaccctga gggtacctgg agatataata agctatgcc
496583DNAClostridium tyrobutyricum 65ccaacaaatt cagaatttat
agctatggtt gcatttaaat acatctcatg ttaaggttca 60acttcagccc tcctgtgaaa
ttg 836681DNAClostridium tyrobutyricum 66atagctataa attctgaatt
tgttggttta ccgttgaacc ttaacatgag atgtatttaa 60atcccataga agctctatac
t 816747DNAClostridium tyrobutyricum 67ctgtatccat atgaccatga
ttacgttatg aaagcgatta cctatat 476869DNAClostridium tyrobutyricum
68gcaagaaaac ggtatattta aatacatctc atgttaaggt tcaacaatat tcctcctaaa
60tttataatc 696969DNAClostridium tyrobutyricum 69ggttcaacaa
aacggtatat ttaaatacat ctcatgttaa ggttcaactt cagccctcct 60gtgaaattg
697067DNAClostridium tyrobutyricum 70atttaaatat accgttttgt
tgaaccttaa catgagatgt atttaaatcc catagaagct 60ctatact
677175DNAClostridium tyrobutyricum 71caactgagag caagaaaacg
gtatatttaa atacatctca tgttaaggtt caacttcagc 60cctcctgtga aattg
757273DNAClostridium tyrobutyricum 72aaatataccg ttttcttgct
ctcagttgaa ccttaacatg agatgtattt aaatcccata 60gaagctctat act
737381DNAClostridium tyrobutyricum 73ctaatagtag tgagagcaag
aaaacggtat atttaaatac atctcatgtt aaggttcaac 60ttcagccctc ctgtgaaatt
g 817479DNAClostridium tyrobutyricum 74ataccgtttt cttgctctca
ctactattag gttgaacctt aacatgagat gtatttaaat 60cccatagaag ctctatact
797588DNAClostridium tyrobutyricum 75gatatagcta atagtagtga
gagcaagaaa acggtatatt taaatacatc tcatgttaag 60gttcaacttc agccctcctg
tgaaattg 887688DNAClostridium tyrobutyricum 76ttgctctcac tactattagc
tatatcatta ttaaacattg ttgaacctta acatgagatg 60tatttaaatc ccatagaagc
tctatact 887750DNAClostridium tyrobutyricum 77catgattacg aattcgagct
cggtaccgtt caaggtatga gtggaagtcc 507855DNAClostridium tyrobutyricum
78tggacgcgtg acgtcgactc tagagacatc ttctatatat ctgcaaaata gcttc
557950DNAClostridium tyrobutyricum 79cctgactcta gagtcgacgt
cacgcgtcga ttgggccctt atatacttgg 508081DNAClostridium tyrobutyricum
80aggagtattt gtccttataa gaacatccaa atttaaatac atctcatgtt aaggttcaac
60ttcagccctc ctgtgaaatt g 818179DNAClostridium tyrobutyricum
81ttggatgttc ttataaggac aaatactcct gttgaacctt aacatgagat gtatttaaat
60cccatagaag ctctatact 798250DNAClostridium tyrobutyricum
82cgacgttgta aaacgacggc cagtgccatg gagatataat aagctatgcc
508348DNAClostridium tyrobutyricum 83ctgtatccat atgaccatga
ttacggctat attgggtttc atagatcc 488444DNAClostridium tyrobutyricum
84gcacactctg catagtctgt gtaagtatcc aggcctacac atac
448544DNAClostridium tyrobutyricum 85gtatgtgtag gcctggatac
ttacacagac tatgcagagt gtgc 448647DNAClostridium tyrobutyricum
86tggacgcgtg acgtcgactc tagagtagtt ccatttccaa ctacctg
478753DNAClostridium tyrobutyricum 87ctgtatccat atgaccatga
ttacgcccgg ggattgggcc cttatatact tgg 538880DNAClostridium
tyrobutyricum 88ggagtatttg tccttataag aacatccaaa tttaaataca
tctcatgtta aggttcaact 60tcagccctcc tgtgaaattg 808979DNAClostridium
tyrobutyricum 89ggatgttctt ataaggacaa atactcctgt tgaaccttaa
catgagatgt atttaaatat 60accgttttct tgctctcac 799049DNAClostridium
tyrobutyricum 90ctacaactaa tacaccctga gggtacctgg agatataata
agctatgcc 499150DNAClostridium tyrobutyricum 91catgattacg
aattcgagct cggtaccgct atattgggtt tcatagatcc 509245DNAClostridium
tyrobutyricum 92ggacttccac tcataccttg aactagttcc atttccaact acctg
459345DNAClostridium tyrobutyricum 93caggtagttg gaaatggaac
tagttcaagg tatgagtgga agtcc 459455DNAClostridium tyrobutyricum
94tggacgcgtg acgtcgactc tagagacatc ttctatatat ctgcaaaata gcttc
559547DNAClostridium tyrobutyricum 95ctgtatccat atgaccatga
ttacggattg ggcccttata tacttgg 479681DNAClostridium tyrobutyricum
96agtattaggt acaggattta cagcacaatc atttaaatac atctcatgtt aaggttcaac
60ttcagccctc ctgtgaaatt g 819779DNAClostridium tyrobutyricum
97gtgctgtaaa tcctgtacct aatactgaac gttgaacctt aacatgagat gtatttaaat
60cccatagaag ctctatact 799849DNAClostridium tyrobutyricum
98gtcgactcta gaggatcccc gggtacctgg agatataata agctatgcc
499951DNAClostridium tyrobutyricum 99ggcatagctt attatatctc
caggtacgta tcaactacgc ctaaattctc c 5110048DNAClostridium
tyrobutyricum 100taggctgttc agggatcccc gggtaccttt cgtttctccc
ttcaagat 4810148DNAClostridium tyrobutyricum 101ggagaaacga
aaggtacccg gggatccctg aacagcctat ggaagacc 4810246DNAClostridium
tyrobutyricum 102tggacgcgtg acgtcgactc tagagcaccg tcaattgcac atacac
4610349DNAClostridium tyrobutyricum 103tatcttgaag ggagaaacga
aaggtacatg aaagtcacaa cagtaaagg 4910451DNAClostridium tyrobutyricum
104ttatggtctt ccataggctg ttcagggttg aaatatgaag gtttaaggtt g
5110549DNAClostridium tyrobutyricum 105tatcttgaag ggagaaacga
aaggtacatg aaagttacaa atcaaaaag 4910651DNAClostridium tyrobutyricum
106ttatggtctt ccataggctg ttcaggttaa aatgatttta tatagatatc c
5110747DNAClostridium tyrobutyricum 107ctgtatccat atgaccatga
ttacggattg ggcccttata tacttgg 4710880DNAClostridium tyrobutyricum
108agttgtaggg ttgatccatc ttctacaaga tttaaataca tctcatgtta
aggttcaact 60tcagccctcc tgtgaaattg 8010980DNAClostridium
tyrobutyricum 109gtagaagatg gatcaaccct acaacttggt agttgaacct
taacatgaga tgtatttaaa 60tcccatagaa gctctatact 8011049DNAClostridium
tyrobutyricum 110gtcgactcta gaggatcccc gggtacctgg agatataata
agctatgcc 4911148DNAClostridium tyrobutyricum 111ggcatagctt
attatatctc caggtacacc catgctgcaa agcaagtt 4811249DNAClostridium
tyrobutyricum 112tgagaaagct aaggatcccc gggtaccaaa aaccaccctt
tcataaatt 4911349DNAClostridium tyrobutyricum 113gggtggtttt
tggtacccgg ggatccttag ctttctcaaa agatatttt 4911446DNAClostridium
tyrobutyricum 114tggacgcgtg acgtcgactc tagagccata tgcggtggtt atcaac
4611549DNAClostridium tyrobutyricum 115aatttatgaa agggtggttt
ttggtacatg aaagtcacaa cagtaaagg 4911652DNAClostridium tyrobutyricum
116ttaaaaatat cttttgagaa agctaaggtt gaaatatgaa ggtttaaggt tg
5211749DNAClostridium tyrobutyricum 117aatttatgaa agggtggttt
ttggtacatg aaagttacaa atcaaaaag 4911852DNAClostridium tyrobutyricum
118ttaaaaatat cttttgagaa agctaagtta aaatgatttt atatagatat cc
5211946DNAClostridium tyrobutyricum 119ctgtatccat atgaccatga
ttacggtaga ctttaaggat ggaacc 4612080DNAClostridium tyrobutyricum
120tcgactctag aggatccccg ggtaccgaat tctgtcgact gcgatgagct
aggtcagtaa 60aaaccaccct ttcataaatt 8012148DNAClostridium
tyrobutyricum 121atataattta tgaaagggtg gtttttatga aagtcacaac
agtaaagg 4812256DNAClostridium tyrobutyricum 122cgactctaga
ggatccccgg gtaccgaatt cgttgaaata tgaaggttta aggttg
5612348DNAClostridium tyrobutyricum 123atataattta tgaaagggtg
gtttttatga aagttacaaa tcaaaaag 4812457DNAClostridium tyrobutyricum
124cgactctaga ggatccccgg gtaccggtaa ccttaaaatg attttatata gatatcc
5712521DNAClostridium tyrobutyricum 125tgttcctgta ggatcagtat c
2112621DNAClostridium tyrobutyricum 126ggactgtacc tctggtagtt c
2112723DNAClostridium tyrobutyricum 127gttgaaagac agctatatct tgg
2312821DNAClostridium tyrobutyricum 128atgccatgtg attctccata g
2112923DNAClostridium tyrobutyricum 129tctatacctt cagatacttc tgg
2313022DNAClostridium tyrobutyricum 130ctcacctcta tacattagcc ac
2213123DNAClostridium tyrobutyricum 131gccattaagt acaaatgaga tag
2313223DNAClostridium tyrobutyricum 132gccattaagt acaaatgaga tag
23133862PRTClostridium acetobutylicum 133Met Lys Val Thr Thr Val
Lys Glu
Leu Asp Glu Lys Leu Lys Val Ile1 5 10 15Lys Glu Ala Gln Lys Lys Phe
Ser Cys Tyr Ser Gln Glu Met Val Asp 20 25 30Glu Ile Phe Arg Asn Ala
Ala Met Ala Ala Ile Asp Ala Arg Ile Glu 35 40 45Leu Ala Lys Ala Ala
Val Leu Glu Thr Gly Met Gly Leu Val Glu Asp 50 55 60Lys Val Ile Lys
Asn His Phe Ala Gly Glu Tyr Ile Tyr Asn Lys Tyr65 70 75 80Lys Asp
Glu Lys Thr Cys Gly Ile Ile Glu Arg Asn Glu Pro Tyr Gly 85 90 95Ile
Thr Lys Ile Ala Glu Pro Ile Gly Val Val Ala Ala Ile Ile Pro 100 105
110Val Thr Asn Pro Thr Ser Thr Thr Ile Phe Lys Ser Leu Ile Ser Leu
115 120 125Lys Thr Arg Asn Gly Ile Phe Phe Ser Pro His Pro Arg Ala
Lys Lys 130 135 140Ser Thr Ile Leu Ala Ala Lys Thr Ile Leu Asp Ala
Ala Val Lys Ser145 150 155 160Gly Ala Pro Glu Asn Ile Ile Gly Trp
Ile Asp Glu Pro Ser Ile Glu 165 170 175Leu Thr Gln Tyr Leu Met Gln
Lys Ala Asp Ile Thr Leu Ala Thr Gly 180 185 190Gly Pro Ser Leu Val
Lys Ser Ala Tyr Ser Ser Gly Lys Pro Ala Ile 195 200 205Gly Val Gly
Pro Gly Asn Thr Pro Val Ile Ile Asp Glu Ser Ala His 210 215 220Ile
Lys Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn225 230
235 240Gly Val Ile Cys Ala Ser Glu Gln Ser Val Ile Val Leu Lys Ser
Ile 245 250 255Tyr Asn Lys Val Lys Asp Glu Phe Gln Glu Arg Gly Ala
Tyr Ile Ile 260 265 270Lys Lys Asn Glu Leu Asp Lys Val Arg Glu Val
Ile Phe Lys Asp Gly 275 280 285Ser Val Asn Pro Lys Ile Val Gly Gln
Ser Ala Tyr Thr Ile Ala Ala 290 295 300Met Ala Gly Ile Lys Val Pro
Lys Thr Thr Arg Ile Leu Ile Gly Glu305 310 315 320Val Thr Ser Leu
Gly Glu Glu Glu Pro Phe Ala His Glu Lys Leu Ser 325 330 335Pro Val
Leu Ala Met Tyr Glu Ala Asp Asn Phe Asp Asp Ala Leu Lys 340 345
350Lys Ala Val Thr Leu Ile Asn Leu Gly Gly Leu Gly His Thr Ser Gly
355 360 365Ile Tyr Ala Asp Glu Ile Lys Ala Arg Asp Lys Ile Asp Arg
Phe Ser 370 375 380Ser Ala Met Lys Thr Val Arg Thr Phe Val Asn Ile
Pro Thr Ser Gln385 390 395 400Gly Ala Ser Gly Asp Leu Tyr Asn Phe
Arg Ile Pro Pro Ser Phe Thr 405 410 415Leu Gly Cys Gly Phe Trp Gly
Gly Asn Ser Val Ser Glu Asn Val Gly 420 425 430Pro Lys His Leu Leu
Asn Ile Lys Thr Val Ala Glu Arg Arg Glu Asn 435 440 445Met Leu Trp
Phe Arg Val Pro His Lys Val Tyr Phe Lys Phe Gly Cys 450 455 460Leu
Gln Phe Ala Leu Lys Asp Leu Lys Asp Leu Lys Lys Lys Arg Ala465 470
475 480Phe Ile Val Thr Asp Ser Asp Pro Tyr Asn Leu Asn Tyr Val Asp
Ser 485 490 495Ile Ile Lys Ile Leu Glu His Leu Asp Ile Asp Phe Lys
Val Phe Asn 500 505 510Lys Val Gly Arg Glu Ala Asp Leu Lys Thr Ile
Lys Lys Ala Thr Glu 515 520 525Glu Met Ser Ser Phe Met Pro Asp Thr
Ile Ile Ala Leu Gly Gly Thr 530 535 540Pro Glu Met Ser Ser Ala Lys
Leu Met Trp Val Leu Tyr Glu His Pro545 550 555 560Glu Val Lys Phe
Glu Asp Leu Ala Ile Lys Phe Met Asp Ile Arg Lys 565 570 575Arg Ile
Tyr Thr Phe Pro Lys Leu Gly Lys Lys Ala Met Leu Val Ala 580 585
590Ile Thr Thr Ser Ala Gly Ser Gly Ser Glu Val Thr Pro Phe Ala Leu
595 600 605Val Thr Asp Asn Asn Thr Gly Asn Lys Tyr Met Leu Ala Asp
Tyr Glu 610 615 620Met Thr Pro Asn Met Ala Ile Val Asp Ala Glu Leu
Met Met Lys Met625 630 635 640Pro Lys Gly Leu Thr Ala Tyr Ser Gly
Ile Asp Ala Leu Val Asn Ser 645 650 655Ile Glu Ala Tyr Thr Ser Val
Tyr Ala Ser Glu Tyr Thr Asn Gly Leu 660 665 670Ala Leu Glu Ala Ile
Arg Leu Ile Phe Lys Tyr Leu Pro Glu Ala Tyr 675 680 685Lys Asn Gly
Arg Thr Asn Glu Lys Ala Arg Glu Lys Met Ala His Ala 690 695 700Ser
Thr Met Ala Gly Met Ala Ser Ala Asn Ala Phe Leu Gly Leu Cys705 710
715 720His Ser Met Ala Ile Lys Leu Ser Ser Glu His Asn Ile Pro Ser
Gly 725 730 735Ile Ala Asn Ala Leu Leu Ile Glu Glu Val Ile Lys Phe
Asn Ala Val 740 745 750Asp Asn Pro Val Lys Gln Ala Pro Cys Pro Gln
Tyr Lys Tyr Pro Asn 755 760 765Thr Ile Phe Arg Tyr Ala Arg Ile Ala
Asp Tyr Ile Lys Leu Gly Gly 770 775 780Asn Thr Asp Glu Glu Lys Val
Asp Leu Leu Ile Asn Lys Ile His Glu785 790 795 800Leu Lys Lys Ala
Leu Asn Ile Pro Thr Ser Ile Lys Asp Ala Gly Val 805 810 815Leu Glu
Glu Asn Phe Tyr Ser Ser Leu Asp Arg Ile Ser Glu Leu Ala 820 825
830Leu Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Phe Pro Leu Thr Ser
835 840 845Glu Ile Lys Glu Met Tyr Ile Asn Cys Phe Lys Lys Gln Pro
850 855 860134858PRTClostridium acetobutylicum 134Met Lys Val Thr
Asn Gln Lys Glu Leu Lys Gln Lys Leu Asn Glu Leu1 5 10 15Arg Glu Ala
Gln Lys Lys Phe Ala Thr Tyr Thr Gln Glu Gln Val Asp 20 25 30Lys Ile
Phe Lys Gln Cys Ala Ile Ala Ala Ala Lys Glu Arg Ile Asn 35 40 45Leu
Ala Lys Leu Ala Val Glu Glu Thr Gly Ile Gly Leu Val Glu Asp 50 55
60Lys Ile Ile Lys Asn His Phe Ala Ala Glu Tyr Ile Tyr Asn Lys Tyr65
70 75 80Lys Asn Glu Lys Thr Cys Gly Ile Ile Asp His Asp Asp Ser Leu
Gly 85 90 95Ile Thr Lys Val Ala Glu Pro Ile Gly Ile Val Ala Ala Ile
Val Pro 100 105 110Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser
Leu Ile Ser Leu 115 120 125Lys Thr Arg Asn Ala Ile Phe Phe Ser Pro
His Pro Arg Ala Lys Lys 130 135 140Ser Thr Ile Ala Ala Ala Lys Leu
Ile Leu Asp Ala Ala Val Lys Ala145 150 155 160Gly Ala Pro Lys Asn
Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu 165 170 175Leu Ser Gln
Asp Leu Met Ser Glu Ala Asp Ile Ile Leu Ala Thr Gly 180 185 190Gly
Pro Ser Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro Ala Ile 195 200
205Gly Val Gly Ala Gly Asn Thr Pro Ala Ile Ile Asp Glu Ser Ala Asp
210 215 220Ile Asp Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr
Asp Asn225 230 235 240Gly Val Ile Cys Ala Ser Glu Gln Ser Ile Leu
Val Met Asn Ser Ile 245 250 255Tyr Glu Lys Val Lys Glu Glu Phe Val
Lys Arg Gly Ser Tyr Ile Leu 260 265 270Asn Gln Asn Glu Ile Ala Lys
Ile Lys Glu Thr Met Phe Lys Asn Gly 275 280 285Ala Ile Asn Ala Asp
Ile Val Gly Lys Ser Ala Tyr Ile Ile Ala Lys 290 295 300Met Ala Gly
Ile Glu Val Pro Gln Thr Thr Lys Ile Leu Ile Gly Glu305 310 315
320Val Gln Ser Val Glu Lys Ser Glu Leu Phe Ser His Glu Lys Leu Ser
325 330 335Pro Val Leu Ala Met Tyr Lys Val Lys Asp Phe Asp Glu Ala
Leu Lys 340 345 350Lys Ala Gln Arg Leu Ile Glu Leu Gly Gly Ser Gly
His Thr Ser Ser 355 360 365Leu Tyr Ile Asp Ser Gln Asn Asn Lys Asp
Lys Val Lys Glu Phe Gly 370 375 380Leu Ala Met Lys Thr Ser Arg Thr
Phe Ile Asn Met Pro Ser Ser Gln385 390 395 400Gly Ala Ser Gly Asp
Leu Tyr Asn Phe Ala Ile Ala Pro Ser Phe Thr 405 410 415Leu Gly Cys
Gly Thr Trp Gly Gly Asn Ser Val Ser Gln Asn Val Glu 420 425 430Pro
Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu Arg Arg Glu Asn 435 440
445Met Leu Trp Phe Lys Val Pro Gln Lys Ile Tyr Phe Lys Tyr Gly Cys
450 455 460Leu Arg Phe Ala Leu Lys Glu Leu Lys Asp Met Asn Lys Lys
Arg Ala465 470 475 480Phe Ile Val Thr Asp Lys Asp Leu Phe Lys Leu
Gly Tyr Val Asn Lys 485 490 495Ile Thr Lys Val Leu Asp Glu Ile Asp
Ile Lys Tyr Ser Ile Phe Thr 500 505 510Asp Ile Lys Ser Asp Pro Thr
Ile Asp Ser Val Lys Lys Gly Ala Lys 515 520 525Glu Met Leu Asn Phe
Glu Pro Asp Thr Ile Ile Ser Ile Gly Gly Gly 530 535 540Ser Pro Met
Asp Ala Ala Lys Val Met His Leu Leu Tyr Glu Tyr Pro545 550 555
560Glu Ala Glu Ile Glu Asn Leu Ala Ile Asn Phe Met Asp Ile Arg Lys
565 570 575Arg Ile Cys Asn Phe Pro Lys Leu Gly Thr Lys Ala Ile Ser
Val Ala 580 585 590Ile Pro Thr Thr Ala Gly Thr Gly Ser Glu Ala Thr
Pro Phe Ala Val 595 600 605Ile Thr Asn Asp Glu Thr Gly Met Lys Tyr
Pro Leu Thr Ser Tyr Glu 610 615 620Leu Thr Pro Asn Met Ala Ile Ile
Asp Thr Glu Leu Met Leu Asn Met625 630 635 640Pro Arg Lys Leu Thr
Ala Ala Thr Gly Ile Asp Ala Leu Val His Ala 645 650 655Ile Glu Ala
Tyr Val Ser Val Met Ala Thr Asp Tyr Thr Asp Glu Leu 660 665 670Ala
Leu Arg Ala Ile Lys Met Ile Phe Lys Tyr Leu Pro Arg Ala Tyr 675 680
685Lys Asn Gly Thr Asn Asp Ile Glu Ala Arg Glu Lys Met Ala His Ala
690 695 700Ser Asn Ile Ala Gly Met Ala Phe Ala Asn Ala Phe Leu Gly
Val Cys705 710 715 720His Ser Met Ala His Lys Leu Gly Ala Met His
His Val Pro His Gly 725 730 735Ile Ala Cys Ala Val Leu Ile Glu Glu
Val Ile Lys Tyr Asn Ala Thr 740 745 750Asp Cys Pro Thr Lys Gln Thr
Ala Phe Pro Gln Tyr Lys Ser Pro Asn 755 760 765Ala Lys Arg Lys Tyr
Ala Glu Ile Ala Glu Tyr Leu Asn Leu Lys Gly 770 775 780Thr Ser Asp
Thr Glu Lys Val Thr Ala Leu Ile Glu Ala Ile Ser Lys785 790 795
800Leu Lys Ile Asp Leu Ser Ile Pro Gln Asn Ile Ser Ala Ala Gly Ile
805 810 815Asn Lys Lys Asp Phe Tyr Asn Thr Leu Asp Lys Met Ser Glu
Leu Ala 820 825 830Phe Asp Asp Gln Cys Thr Thr Ala Asn Pro Arg Tyr
Pro Leu Ile Ser 835 840 845Glu Leu Lys Asp Ile Tyr Ile Lys Ser Phe
850 855
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